Integrated Nucleic Acid Analysis

ABSTRACT

The present disclosure provides fully integrated microfluidic systems to perform nucleic acid analysis. These processes include sample collection, nucleic acid extraction and purification, amplification, sequencing, and separation and detection. The present disclosure also provides optical detection systems and methods for separation and detection of biological molecules. In particular, the various aspects of the invention enable the simultaneous separation and detection of a plurality of biological molecules, typically fluorescent dye-labeled nucleic acids, within one or a plurality of microfluidic chambers or channels. The nucleic acids can be labeled with at least 6 dyes, each having a unique peak emission wavelength. The present systems and methods are particularly useful for DNA fragment sizing applications such as human identification by genetic fingerprinting and DNA sequencing applications such as clinical diagnostics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/191,952 filed on27 Jul. 2011 which is a continuation of U.S. Ser. No. 12/080,751, filedon 4 Apr. 2008, now issued as U.S. Pat. No. 8,018,593 and entitled“Integrated Nucleic Acid Analysis”, which claims the benefit of thefiling date, under 35 U.S.C. § 119(e), of U.S. Provisional ApplicationSer. No. 60/921,802 filed 4 Apr. 2007; U.S. Provisional Application Ser.No. 60/964,502 filed 13 Aug. 2007; and U.S. Provisional Application Ser.No. 61/028,073 filed 12 Feb. 2008, each of that is hereby incorporatedby reference in its entirety. This application also incorporates byreference, in their entireties, two U.S. Patent applications filed onApr. 4, 2008; the first entitled “METHODS FOR RAPID MULTIPLEXEDAMPLIFICATION OF TARGET NUCLEIC ACIDS”, U.S. Ser. No. 12/080,746, nowissued as U.S. Pat. No.8,425,861; and the second entitled “PLASTICMICROFLUIDIC SEPARATION AND DETECTION PLATFORMS”, U.S. Ser. No.12/080,745, now issued as U.S. Pat. No. 8,858,770.

FIELD OF THE INVENTION

This invention is in the field of microfluidics for the analysis ofnucleic acids.

BACKGROUND OF THE INVENTION

There is an unmet need for the development of instruments andtechnologies that would permit fully integrated (i.e., sample-in toresults-out) focused nucleic acid analysis, defined as the rapididentification (by nucleic acid sequencing or fragment sizing) of asubset of a given human, animal, plant, or pathogen genome. Focusednucleic acid sequencing will enable end-users to make real-timeclinical, forensic, or other decisions. For example, many common humandiseases can be diagnosed based on less than 1000 base pairs of DNAsequence, orders of magnitude less than required to generate a completehuman genome. Similarly, precise determination of the sizes of sets ofless than 20 specific DNA fragments generated by short tandem repeatanalysis is sufficient to identify a given individual. Depending on theapplication, focused nucleic analysis can be performed in a variety ofsettings, including hospital laboratories, physician's offices, thebedside, or, in the case of forensic or environmental applications, inthe field.

There are several unmet needs for improved DNA sequencing and fragmentsizing systems. First, there is an unmet need for DNA sequencing andfragment sizing instruments that are easy to use and do not requirehighly trained operators. Second, there is an unmet need for systemsthat eliminate all manual processing. As a result, only minimal operatortraining would be required and the system could be readily operated byindividuals constrained by challenging environments such as would beencountered, for example, by a first responder wearing a haz-mat suit.

Third, there is an unmet need for ultrafast analysis that does notsacrifice the need for complete, accurate, and reliable data. For humanidentification applications, an appropriate time to result is 45 minutesor less, well under the days to weeks required using conventionaltechnology. For clinical applications such as sequencing infectiousagents to determine an appropriate treatment regimen, 90 minutes or lessis a reasonable time to answer, allowing treatment with antibacterialand antiviral medications to be initiated shortly after a patient'sarrival in an emergency room. Regardless of application, there is anunmet need to generate actionable data in real time. A short time toanswer also allows a concomitant increase in sample throughput.

Fourth, there is an unmet need for miniaturization. Many DNA analysissystems require an entire laboratory and related support. For example,the high throughput Genome Sequencer FLX (Roche Diagnostics Corp,Indianapolis, Ind.) DNA sequencing system requires only a benchtop forinstallation but a large laboratory to perform the required libraryconstruction. Miniaturization is important both for laboratory andpoint-of-care use as well as field operation. It is also important forcost reduction per sample.

Fifth, there is an unmet need for ruggedization. For many applications,particularly those in forensics, the military, and homeland defense, theDNA analysis instrument must be operable in the field. Accordingly, theinstrument must be capable of transport whether carried on a soldier'sback, driven in a police vehicle, or dropped from a helicopter into abattlefield. Similarly, the instrument must be able to withstand andfunction under environmental extremes, including temperature, humidityand airborne particulates (e.g., sand).

Sixth, there is an unmet need for systems that can accept multiplesample types and perform highly multiplexed analyses in parallel. Formost applications, capability of analysis of DNA from a single sampletype in a singleplex reaction is not acceptable to perform meaningfulDNA analysis.

Developers of microfluidics (also referred to as micro total analysissystems (μTAS) or lab-on-a-chip technologies, see, Manz et al., Sens.Actuators B 1990, 1, 244-248) who are seeking to condense complex seriesof laboratory manipulations onto biochips have recognized certain ofthese unmet needs, but to date, have been unable to design integratedbiochips and instruments that perform all of the biochemical andphysical processes necessary or desirable to allow microfluidic nucleicacid analysis to address these needs. As a result, focused nucleic acidanalysis has not entered into widespread use in society today.

The development of microfluidic systems involves the integration ofmicrofabricated components, such as microscale separations, reactions,microvalves and pumps and various detection schemes into fullyfunctional devices (see, e.g., Pal et al., Lab Chip 2005, 5, 1024-1032).Since Manz et al. (supra), demonstrated capillary electrophoresis on achip in the early 1990's, others have sought to improve upon it. Severalgroups have demonstrated integration of DNA processing functionalitywith biochip separation and detection. Integrated devices in aglass-PDMS (polydimethylsiloxane) hybrid structure have been reported(Blazej et al., Proc Natl Acad Sci USA 2006, 103, 7240-5; Easley et al.,Proc. Natl. Acad. Sci. USA 2006, 103, 19272-7; and Liu et al., Anal.Chem. 2007, 79, 1881-9). Liu coupled multiplex polymerase chain reaction(PCR), separation and four dye detection for human identification byshort tandem repeat (STR) sizing. Blazej coupled Sanger sequencingreaction, Sanger reaction cleanup, electrophoretic separation and fourdye detection for DNA sequencing of pUC18 amplicon. Easley coupled solidphase extraction of DNA, PCR, electrophoretic separation and singlecolor detection to identify the presence of bacterial infection inblood. An integrated silicon-glass device coupling PCR, electrophoreticseparation and single color detection was demonstrated by Burns (Pal,2005, Id.). A hybrid device coupling a glass-PDMS portion for PCR to apoly(methyl methacrylate) (PMMA) portion for electrophoretic separationand single color detection for identifying the presence of bacteria DNAwas reported by Huang (Huang et al., Electrophoresis 2006, 27,3297-305).

Koh et al., report a plastic device that coupled PCR to biochipelectrophoretic separation and single color detection for identifyingthe presence of bacterial DNA (Koh et al., Anal. Chem. 2003, 75,4591-8). A silicone based device that couples DNA extraction, PCRamplification, biochip electrophoretic separation and single colordetection was reported by Asogawa (Asogowa M, Development of portableand rapid human DNA Analysis System Aiming on-site Screening, 18thInternational Symposium on Human Identification, Poster, Oct. 1-4, 2007,Hollywood, Calif., USA). U.S. Pat. No. 7,332,126 (Tooke et al.)describes the use of centrifugal force to effect microfluidic operationsrequired for nucleic acid isolation and cycle sequencing. However, thisapproach is based on small sample volumes, (those of the order of one toa few μL). As a result, the device is not useful for the processing oflarge samples for the isolation and analysis of nucleic acids,especially in highly parallel fashion, because the fluid samples must beapplied to the device while stationary, which is, the disc must be ableto contain all the fluids required for operation prior to centrifugation(potentially up to 100 s of mL for a highly-parallel device). Secondly,the device is limited to sample preparation and cycle sequencing, ofbacterial clones (e.g., plasmid DNA).

There are several deficiencies in those devices that attempt tointegrate DNA processing with biochip electrophoretic separation. First,detection is limited by either information content per assay (most usesingle color detectors although some have up to four color detectionsystems) or throughput (single sample or two sample capability). Second,these devices do not represent complete sample-to-answer integration,e.g., Blazej's device requires off-board amplification of template DNAprior to cycle sequencing, while others use samples that requirepre-processing of some sort (e.g., Easley and Tooke require lysis of thesample before addition). Third, some of the processing choices made forthese devices negatively impact time-to-answer: for example, thehybridization-based method of Blazej requires more than 20 minutes forcleanup of the cycle sequencing product. Fourth, many of these devicesare fabricated in part or in-whole glass or silicon. The use of thesesubstrates and corresponding fabrication techniques make them inherentlycostly (Gardeniers et al., Lab-on-a-Chip (Oosterbroeck R E, van den BergA, Eds.). Elsevier: London, pp 37-64 (2003)) and limit them toapplications where reuse of the devices must be performed; for manyapplications (such as human ID) this leads to the risk of samplecontamination. Finally, the demonstrated technology is inappropriate fortwo applications, human identification via STR analysis and sequencing.For example, the Easley and Pal devices both suffer from poorresolution—much worse than a single base. Fragment sizing applications(e.g., human identification by analysis of short tandem repeat profiles)and sequencing both require single base resolution.

In addition to the limitations of the prior art with respect tomicrofluidic integration, problems with respect to fluorescencedetection also limit the widespread application of nucleic acid analysisbeyond conventional laboratory work. The most widely used commercialsequencing kits (BigDye™ v3.1 [Applied Biosystems] and DYEnamic™ ET [GEHealthcare Biosciences Corp, Piscataway, N.J.]) are based on a twentyyear old detection method for four color (see, e.g., U.S. Pat. Nos.4,855,225; 5,332,666; 5,800,996; 5,847162; 5,847,162). This method isbased on resolution of the emission signal of a dye-labeled nucleotideinto four different colors, one representing each of the four bases.These four-color dye systems have several disadvantages, includinginefficient excitation of the fluorescent dyes, significant spectraloverlap, and inefficient collection of the emission signals. The fourcolor dye systems are especially problematic because they limit theamount of information that can be gained from a given electrophoretic(or other) separation of sequenced products.

There is an unmet need for a system capable of achieving highinformation content assays in electrophoretic systems based onseparation and detection of DNA fragments by both fragment size and bycolor (dye wavelengths). The maximum number of DNA fragments that can bedistinguished by electrophoresis is determined by the readlength of theseparation and the resolution of the device. The maximum number ofcolors that can be detected is determined in part by the availability offluorescent dyes and the wavelength discrimination of the detectionsystem. Current biochip detection systems are typically limited tosingle color, although up to 4 color detection has been reported.

STR analysis for human identification is an example of DNA fragmentsizing based on color multiplexing and allows simultaneous analysis upto 16 loci (AmpFISTR Identifiler kit; Applied Biosystems, Foster City,Calif.) and PowerPlex16 kit (Promega Corporation, Madison, Wis.). Usingfour or five fluorescent dyes, a single separation channel candiscriminate among the sizes of the many allelic variants of each locus.Several fragment sizing applications would require more than 16fragments to be separated and detected on a single lane. For example,the identification of pathogens by fingerprinting (i.e., the separationand detection of a large number of characteristic DNA fragments) and thediagnosis of aneuploidy by surveying the entire human genome can beaccomplished by looking at several dozen or several hundred loci,respectively.

One approach to increasing the number of loci that can be detected in asingle separation channel is to broaden the range of fragment sizesgenerated, in part by increasing the fragment sizes of additional loci.The use of longer fragments for additional loci is not ideal, however,as amplification of larger fragments is more susceptible to inhibitorsand DNA degradation, leading to lower yields of longer fragmentsrelative to shorter fragments. Furthermore, the generation of longerfragments also requires an increase in the extension time and hence, anincrease in the total assay time. There is an unmet need to increasingthe number of loci that can be detected in a given separation channel byincreasing the number of dye colors that can be simultaneously detected.

There is an unmet need to increase the capacity of Sanger sequencingseparations (and therefore decrease the cost, labor, and space of theprocess) by increasing the number of DNA sequences that can be analyzedin a single separation channel. In addition, in some applications,multiple DNA fragments are sequenced that generate difficult to read“mixed sequence” data; there is a need to develop an approach in thatmixed sequences can be interpreted correctly.

One approach to increasing the capacity of Sanger separation channelsand developing the ability to interpret mixed sequences is to increasethe number of dye colors utilized in the sequencing reactions. In bothDNA sequencing and fragment sizing, multiple fragments labeled withdifferent dyes can be detected at the same time. In general, theseparation between peak emission wavelengths of adjacent dyes must belarge enough relative to peak width of the dyes. Accordingly, thethroughput of each separation channel can, for example, be doubled byutilizing two sets of 4 dyes in two independent sequencing reactions,and combining the products, and separating them on a single channel.This methodology requires the use of a total of 8 dye colors, with thefirst sequence reaction using a set of 4 dye colors applied to label thedideoxynucleuotide terminators, and the second reaction another set of 4dye colors applied to the label the terminators; each set of dye colorsis independent so that no overlap in interpretation of the two sequencesis possible. Using this same approach, a set of 12 dyes can be utilizedto allow simultaneous analysis of the sequence of three DNA fragments ina single channel, a set of 16 dyes allows the analysis of foursequences, and so on, dramatically increasing the information capacityof Sanger separations.

The novel instruments and biochips of this application satisfy manyunmet needs, including those outlined above.

SUMMARY OF THE INVENTION

This invention provides fully integrated microfluidic systems to performnucleic acid analysis. These processes include sample collection, DNAextraction and purification, amplification (that can be highlymultiplexed), sequencing, and separation and detection of the DNAproducts.

The separation and detection modules of this invention are ruggedizedand capable of better than single base resolution. They are capable ofdetecting six or more colors and as such are useful for generating highinformation content from sequencing and fragment sizing applications.

Highly multiplexed rapid PCR on biochips is the subject of an U.S.Patent Application, filed on Apr. 4, 2008, assigned U.S. Ser. No.12/080,746, and entitled, “METHODS FOR RAPID MULTIPLEXED AMPLIFICATIONOF TARGET NUCLEIC ACIDS;” it is expressly hereby incorporated byreference in its entirety. Further, the PCR products can be separatedand detected within a biochip as described in the U.S. patentapplication entitled, “PLASTIC MICROFLUIDIC SEPARATION AND DETECTIONPLATFORMS”, and assigned U.S. Ser. No. 12/080,745, which is expresslyhereby incorporated by reference in its entirety.

Accordingly, in a first aspect, the invention provides optical detectorscomprising one or more light sources positioned for illuminating one ora plurality of detection positions on a substrate; one or a plurality offirst optical elements positioned for collecting and directing lightemanating from the detection positions on the substrate; and a lightdetector positioned to accept light from the first optical elements,wherein the light detector comprises a wavelength dispersive element toseparate the light from the first optical elements according to lightwavelength and positioned to provide a portion of the separated light tothe detection elements, wherein each of the detection elements are incommunication with a first control element for simultaneously collectingdetection information from each of the detection elements and, whereinsaid light detector detects fluorescence from at least 6 dyes labeled toone or more biological molecules, each dye having a unique peak emissionwavelength.

In a second aspect, the invention provides systems for separation anddetection of biological molecules comprising, a component forsimultaneously separating a plurality of biological molecules in one ora plurality of channels on a substrate, wherein each channel comprises adetection position; one or more light sources positioned to illuminatethe detection positions on the substrate; one or a plurality of firstoptical elements positioned for collecting and directing light emanatingfrom the detection positions; and a light detector positioned to acceptlight directed from the first optical elements, wherein the lightdetector comprises a wavelength dispersive element to separate the lightfrom the first optical elements according to light wavelength andpositioned to provide a portion of the separated light to the detectionelements, wherein each of the detection elements are in communicationwith a first control element for simultaneously collecting detectioninformation from each of the detection elements and, wherein said lightdetector detects fluorescence from at least 6 dyes labeled to one ormore biological molecules, each dye having a unique peak wavelength.

In a third aspect, the invention provides methods for separating anddetecting a plurality of biological molecules comprising, providing oneor a plurality of analysis samples into one or a plurality ofmicrofluidic channels on a substrate, wherein each microfluidic channelcomprises a detection position, and each analysis sample independentlycomprises a plurality of biological molecules, each independentlylabeled with one of at least 6 dyes, each dye having a unique peakwavelength ; simultaneously separating the plurality of labeledbiological molecules in each microfluidic channel; and detecting theplurality of separated target analytes in each microfluidic channel by,illuminating each detection position with a light source; collecting thelight emanating from each detection position; directing the collectedlight to a light detector; and (i) separating the collected light bylight wavelength; and (ii) simultaneously detecting the fluorescencefrom at least 6 dyes labeled to one or more biological molecules, eachdye having a unique peak wavelength.

In a fourth aspect, the invention provides integrated biochip systemscomprising (a) a biochip comprising one or a plurality microfluidicsystems, wherein each microfluidic system comprises a first reactionchamber in microfluidic communication with a separation chamber, whereinthe first reaction chamber is adapted for nucleic acid extraction;nucleic acid purification; pre-nucleic acid amplification cleanup;nucleic acid amplification; post-nucleic acid amplification cleanup;pre-nucleic acid sequencing cleanup; nucleic acid sequencing;post-nucleic acid sequencing cleanup; reverse transcription; pre-reversetranscription cleanup; post-reverse transcription cleanup; nucleic acidligation; nucleic acid hybridization; or quantification; and theseparation chamber comprises a detection position; and (b) a separationand detection system comprising, (i) a separation element forsimultaneously separating a plurality of target analytes in theseparation chambers; (ii) one or more light sources positioned toilluminate the detection positions on the biochip; (iii) one or aplurality of first optical elements positioned for collecting anddirecting light emanating from the detection positions; and (iv) a lightdetector positioned to accept light directed from the first opticalelements, wherein the light detector comprises a wavelength dispersiveelement to separate the light from the first optical elements accordingto light wavelength and positioned to provide a portion of the separatedlight to at least six detection elements, wherein each of the detectionelements are in communication with a first control element forsimultaneously collecting detection information from each of thedetection elements; and wherein said light detector detects fluorescencefrom at least 6 dyes labeled to one or more biological molecules, eachdye having a unique peak wavelength.

In a fifth aspect, the invention provides integrated biochip systemsbiochip system comprising (a) a biochip comprising one or a pluralitymicrofluidic systems, wherein each microfluidic system comprises a firstreaction chamber in microfluidic communication with a separationchamber, wherein the first reaction chamber is adapted for nucleic acidextraction; nucleic acid purification; pre-nucleic acid amplificationcleanup; nucleic acid amplification; post-nucleic acid amplificationcleanup; pre-nucleic acid sequencing cleanup; nucleic acid sequencing;post-nucleic acid sequencing cleanup; reverse transcription; pre-reversetranscription cleanup; post-reverse transcription cleanup; nucleic acidligation; nucleic acid hybridization; or quantification; and theseparation chamber comprises a detection position; and (b) a separationand detection system comprising, (i) a separation element forsimultaneously separating a plurality of biological molecules comprisingDNA sequences, in the separation chambers; (ii) one or more lightsources positioned to illuminate the detection positions on the biochip;(iii) one or a plurality of first optical elements positioned forcollecting and directing light emanating from the detection positions;and (iv) a light detector positioned to accept light directed from thefirst optical elements, wherein the light detector comprises awavelength dispersive element to separate the light from the firstoptical elements according to light wavelength and positioned to providea portion of the separated light to at least six detection elements,wherein each of the detection elements are in communication with a firstcontrol element for simultaneously collecting detection information fromeach of the detection elements; and wherein said light detector detectsfluorescence from at least 8 dyes labeled to one or more DNA sequences,each dye having a unique peak wavelength, said dyes being members of atleast two 4-dye containing subsets, such that said dye sets are capableof detecting at least two DNA sequences in a single channel, wherein thenumber of dyes is a multiple of four, and the number of DNA sequences tobe detected is equal to that multiple, such that each of the differentdyes is present in only one subset.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is an illustration of an embodiment of an integrated biochip forlysis and template amplification for 4 individual samples.

FIG. 2 is an illustration of an embodiment of the first layer of thebiochip of FIG. 1.

FIG. 3 is an illustration of an embodiment of the second layer of thebiochip of FIG. 1.

FIG. 4 is an illustration of an embodiment of the third layer of thebiochip of FIG. 1.

FIG. 5 is an illustration of an embodiment of the fourth layer of thebiochip of FIG. 1.

FIG. 6 is an illustration of an embodiment of assembly and bonding ofthe biochip of FIG. 1.

FIG. 7 is a graph illustrating capillary valving pressure as a functionof inverse hydraulic diameter of the valve for deionized water and cyclesequencing reagents for two valve types, in-plane and through-holevalves.

FIG. 8 is an illustration showing an embodiment of fluidic steps of thebiochip of FIG. 1 for template amplification by PCR.

FIG. 8a is an illustration showing samples and PCR reagents have beenloaded into a biochip of the invention.

FIG. 8b is an illustration showing sample delivery through channels tosample chambers (they are shown at different positions along the samplechannels in order to illustrate the flow path.)

FIG. 8c is an illustration showing the samples in the sample chambers.

FIG. 8d is an illustration showing delivery of PCR reagents to reagentchambers.

FIG. 8e is an illustration showing withdrawal of excess PCR reagent.

FIGS. 8f and 8g are illustrations showing the initial mixing step andretention of the liquids by the first set of capillary valves.

FIGS. 8h through 8j are illustrations showing the mixed liquidsdelivered to the PCR chamber, at that point thermal cycling isinitiated.

FIG. 9 is an illustration showing an embodiment of the fluidic steps ofan integrated biochip.

FIGS. 9a through 9e are illustrations showing the delivery of cyclesequencing reagent to metering chambers in layer 1 and removal of excessreagent from the vicinity of the chambers.

FIGS. 9f and 9g are illustrations showing the introduction of PCRproduct into a Sanger reaction chamber.

FIG. 9h-9k are illustrations showing mixing of Sanger reagent with PCRproduct by reciprocal motion.

FIG. 9l is an illustration showing cycled product in that can be removedfor analysis.

FIG. 10 is a sequencing trace (electropherograms) for sequencing productproduced in the biochip of FIG. 1.

FIG. 11 is an illustration showing an embodiment of an integratedbiochip for the performance of ultrafiltration of a cycle sequencingproduct. The chip assembly is similar to that in biochip 1 except forthe addition of an ultra-filtration (UF) filter 1116 between layers 3and 4.

FIG. 12 is an illustration showing the fluidic steps of the biochip ofFIG. 11 during purification of sequencing product.

FIGS. 12a and 12b are illustrations showing delivery of a Sanger productto the UF input chambers.

FIG. 12c is an illustration showing the sequencing product delivered tothe filtration chamber.

FIG. 12d is an illustration showing is nearly complete filtration of thesequencing product.

FIGS. 12e through 12g are illustrations showing delivery of wash to theUF input chambers and subsequent removal of excess wash from thedelivery channel.

FIG. 12h is an illustration showing the beginning of the first washcycle; it is followed by filtration as in FIG. 12d and a subsequent washcycle.

FIGS. 12i and 12j are illustrations showing elution liquid (the sameliquid as the wash) being delivered to the UF input chambers.

FIGS. 12k through 12m are illustrations showing a single cycle ofpressurizing the UF input chambers with the output ports closed, andthen releasing the pressure to cause reciprocal motion.

FIG. 12n is an illustration showing purified product ready for furtherprocessing or removal.

FIG. 13 is an illustration showing an embodiment of an integratedbiochip for the performance of template amplification, cycle sequencing,sequencing product cleanup, separation by electrophoresis and detectionby laser-induced fluorescence.

FIG. 14 is an illustration showing concentration of labeled nucleic acidfragments by counter electrode and injection into a separation channel.

FIG. 15 is an illustration showing an embodiment of an excitation anddetection system.

FIG. 16 is an illustration showing an embodiment of an excitation anddetection system.

FIG. 17 is an electrophoregram generated for separation and detection ofa 6 dye sample. Each trace in the graph represents the signal from eachof the each of the 32 elements of a 32-anode PMT. Each trace is offsetrelative to each other to allow easy viewing of data.

FIG. 18 is a graph showing the dye spectra of each of the 6 dyes,extracted from the electrophoregram; also shown is the backgroundfluorescence spectra.

FIG. 19 is a graph showing the dye emission spectra for 6-FAM, VIC, NED,PET and LIZ dyes.

FIG. 20 is a graph showing the dye emission spectra for 5-FAM, JOE, NED,and ROX dyes.

FIG. 21 is an electrophoregram generated for separation and detection ofa 4 dye sample. Each trace in the graph represents the signal from eachof the each of the 32 elements of a 32-anode PMT. Each trace is offsetrelative to each other to allow easy viewing of data.

FIG. 22 is a sequencing trace.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS I. Integration andIntegrated Systems A. GENERAL DESCRIPTION OF INTEGRATION

Using microfluidics allows fabrication of features to perform more thanone function on a single biochip. Two or more of these functions can beconnected microfluidically to enable sequential processing of a sample;this coupling is termed integration.

There is a range of possible functions or component processes that mustbe integrated to achieve any given application, though not all processesmust be implemented for any given application. As a result, the chosenintegration methods must be appropriate for effectively connecting anumber of different component processes in different sequences.Processes that can be integrated include, but are not limited to, thefollowing:

-   -   1. Sample insertion;    -   2. Removal of foreign matter (e.g., large particulates such as        dust, fibers)    -   3. Cell separation (i.e., the removal of cells other than those        containing the nucleic acid to be analyzed, such as the removal        of human cells (and accordingly, human genomic DNA) from        clinical samples containing microbial nucleic acids to be        analyzed);    -   4. Concentration of cells containing the nucleic acids of        interest;    -   5. Lysis of cells and extraction of nucleic acids;    -   6. Purification of nucleic acids from the lysate; with possible        concentration of the nucleic acids to smaller volumes;    -   7. Pre-amplification nucleic acid clean-up;    -   8. Post-amplification clean-up;    -   9. Pre-sequencing clean-up;    -   10. Sequencing;    -   11. Post-sequencing clean-up (e.g., to remove unincorporated        dye-labeled terminators and ions that interfere with        electrophoresis;    -   12. Nucleic acid separation;    -   13. Nucleic acid detection;    -   14. Reverse transcription of RNA;    -   15. Pre-reverse transcription clean-up;    -   16. Post-reverse transcription clean-up;    -   17. Nucleic acid ligation;    -   18. Nucleic acid quantification.    -   19. Nucleic acid hybridization; and    -   20. Nucleic acid amplification (e.g., PCR, rolling circle        amplification, strand displacement amplification, and multiple        displacement amplification).        One of many ways in which some of these processes may be        combined is in an integrated system for human identification by        STR analysis. Such an system may require the coupling of DNA        extraction, human specific DNA quantification, addition of a        defined amount of DNA to the PCR reaction, multiplexed PCR        amplification, and separation and detection (optionally,        clean-up steps to remove reaction components or primers can be        incorporated as well). One or more samples can be collected by        techniques such as swabbing (see, Sweet et al., J. Forensic Sci.        1997, 42, 320-2) of whole blood, dried blood, the inner surface        of the cheeks, fingerprints, sexual assault, touch, or other        forensically relevant samples. Exposure to lysate (optionally in        the presence of agitation) releases the DNA from the swab into a        tube.

B. GENERAL DESCRIPTION OF INTEGRATION COMPONENTS AND THEIR USES

1. Sample Collection and Initial Processing

For many applications, the following discrete components areadvantageously integrated into the biochip: sample insertion; removal offoreign matter; removal of interfering nucleic acids; and concentrationof cells of interest. Generally, a pre-processing component of thebiochip accepts samples, performs initial removal of particulates andforeign nucleic acid containing cells, and concentrates the cells ofinterest into small volumes. One approach is to use a sample tube thatcan accept a swab (e.g., resembling a “Q-tip”) and that is filled withlysis solution to perform the lysis and extraction step. The swab can beplaced in contact with a number of cell-containing sites, including abloodstain, a fingerprint, water, an air filter, or a clinical site(e.g., buccal swab, wound swab, nasal swab). The interface of thesetubes with other components of the biochip may include a filter forremoval of foreign matter. Another approach is to use a large-volumeblood or environmental sample acquisition cartridge, which processes a1-100 mL of sample. In the case of blood, a leukocyte reduction mediumcan remove human white blood cells and interfering DNA while passingmicrobes containing nucleic acids of interest. For environmentalsamples, large-mesh filters can be used to remove dust and dirt, whilesmall-mesh filters (e.g., filters of <20 μm, <10 μm, <5 μm, <2.5 μm, <1μm, <0.5 μm, <0.2 μm, <0.1 μm) can be used to trap microbes,concentrating them in a small volume. These pre-processing componentscan be separate consumables or can be attached to the integrated biochipat time of manufacture. Alternatively, the biochip can be designed toperform differential lysis to separate cells by type (e.g., sperm fromvaginal epithelial cells or red blood cells from bacteria).

2. Lysis and Extraction

A variety of lysis and extraction methods can be employed. For example,a typical procedure involves the application of heat after mixing of thesample with a small quantity of a degradative enzyme such asproteinase-K, which breaks down cell walls and releases nucleic acids.Other useful methods are sonication and ultrasonication, either or bothperformed sometimes in the presence of beads.

For example, lysis and extraction can be performed on a samplecontaining 10⁶ cells or less. Depending on the application, a smallernumber of starting cells can be utilized in the biochips and methods ofthe invention, less than 10⁵, less, than 10⁴, less than 10³, less than,10², less than 10, and, in cases when multi-copy sequences are to beanalyzed, less than 1.

3. Purification of Nucleic Acids

One form of nucleic acid purification can be achieved by inserting apurification medium between an input and output channel. Thispurification medium can be silica fiber based and use chaotropic-saltreagents to lyse the biological sample, expose the DNA (and RNA) andbind the DNA (and RNA) to the purification media. The lysate is thentransported via the input channel through the purification medium tobind the nucleic acids. Bound nucleic acid is washed by an ethanol basedbuffer to remove contaminants. This can be accomplished by flowing washreagents via the input channel through the purification membrane. Boundnucleic acid is then eluted from the membrane by flowing an appropriatelow salt buffer (e.g., Boom U.S. Pat. No. 5,234,809). A variation ofthis method involves the use of a differently-configured solid phase.For example, silica gel can be employed to bind nucleic acid.Paramagnetic silica beads can be used, and their magnetic propertiesemployed to immobilize them against a channel or chamber wall duringbinding, wash, and elution steps. Non-magnetized silica beads may alsobe employed, either packed within a tight ‘column’ where they areretained by frits (typically manufactured into the plastic of thedevice, but these may also be inserted during assembly) or “free” duringcertain phases of their operation: Free beads can be mixed with nucleicacids and then flowed against a frit or a weir in the device to trapthem so that they do not interfere with downstream processes. Otherformats include sol-gels with silica particles distributed in the gelmedium and polymer monoliths with silica particle inclusions, in whichthe carrier is cross-linked for greater mechanical stability.Essentially, any nucleic acid purification method that is functional ina conventional setting can be adapted to the integrated biochips of thisinvention.

4. Nucleic Acid Amplification

A variety of nucleic acid amplification methods can be employed, such asPCR and reverse-transcription PCR, which required thermal cyclingbetween at least two, and more typically, three temperatures. Isothermalmethods such as strand displacement amplification can be used, andmultiple displacement amplification can be used for whole genomeamplification. The teachings of the U.S. patent application entitled“METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS”(U.S. Ser. No. 12/080,746) filed on Apr. 4, 2008 is hereby incorporatedby reference in its entirety (supra).

5 Nucleic Acid Quantification

One approach to quantification in a microfluidic format is based uponreal-time PCR. In this method of quantification, a reaction chamber isfabricated between an input and output channel. The reaction chamber iscoupled to a thermal cycler and an optical excitation and detectionsystem is coupled to the reaction chamber to allow fluorescence from thereaction solution to be measured. The amount of DNA in the sample iscorrelated to the intensity of the fluorescence from the reactionchamber per cycle. See, e.g., Heid et al., Genome Research 1996, 6,986-994. Other quantitation methods include the use of intercalatingdyes such as picoGreen, SYBR, or ethidium bromide, either prior to orafter amplification, which may then be detected using eitherfluorescence or absorbance.

6. Secondary Purifications

For STR analysis, multiplex-amplified and labeled PCR product can beused directly for analysis. However, electrophoretic separationperformance can be greatly improved by purification of the product toremove ions necessary for PCR that interfere with the separation orother subsequent steps. Similarly, purification following cyclesequencing or other nucleic acid manipulations can be useful.Collectively, any purification step following the initial extraction orpurification of nucleic acid can be considered a secondary purification.A variety of methods can be employed, including ultrafiltration, in thatsmall ions/primers/unincorporated dye labels are driven through afilter, leaving the desired product on the filter that then can beeluted and applied directly to the separation or subsequent module.Ultrafiltration media include polyethersulfone and regenerated cellulose“woven” filters, as well as track-etch membranes, in which pores ofhighly-uniform size are formed in an extremely thin (1-10 μm) membrane.The latter have the advantage of collecting product of size larger thanthe pore size on the surface of the filter, rather than capturing theproduct at some depth below the surface. The amplified nucleic acids mayalso be purified using the same methods outlined above (i.e., classicsolid phase purification on silica). Still further methods includehydrogels, cross-linked polymers that have the property of pore sizevariability, that is, the pore size changes in response to environmentalvariables such as heat and pH. In one state, the pores are tight and PCRproduct cannot pass through. As the pores dilate, hydrodynamic orelectrophoretic flow of product through the pores is possible. Anothermethod is the use of hybridization, either non-specific hybridization ofproduct to random DNA immobilized on a surface (such as the surface ofbeads) or specific hybridization, in that a complement to a sequence tagon the product is on the solid surface. In this approach, the product ofinterest is immobilized through hybridization and unwanted materialremoved by washing; subsequent heating melts the duplex and releases thepurified product.

7. Cycle Sequencing Reaction

Classic cycle sequencing requires thermal cycling, much as PCR. Thepreferred methods are those employing dye-labeled terminators, such thateach extension product bears a single fluorescent label corresponding tothe final base of the extension reaction.

8. Injection, Separation, and Detection

Injection, separation and detection of labeled nucleic acid fragmentsinto the electrophoresis channel can be performed in a variety of ways,which have been described in the U.S. patent application entitled“PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS”, filed on Apr.4, 2008 and assigned U.S. Ser. No. 12/080,745, which is herebyincorporated by reference in its entirety. First, cross-injectors asdiscussed therein can be used to inject a portion of the sample. In analternative embodiment, electrokinetic injection (“EKI”) can be used. Ineither case, further concentration of sequencing product in the vicinityof the open end of the loading channel (in the case of cross injection)or separation channel (in the case of EKI) can be performed byelectrostatically concentrating product near an electrode. Atwo-electrode sample well on the electrophoresis portion of the chip isshown in FIG. 14. Both electrodes are coated with a permeation layerthat prevents DNA from contact the metal of the electrode but allowsions and water access between the sample well and the electrode. Suchpermeation layers can be formed of cross-linked polyacrylamide (see USPatent Application Publication US 2003-146145-A1). The electrodefarthest from the channel opening is the separation electrode, whilethat nearest the channel opening is the counterelectrode. By chargingthe counterelectrode positively relative to the separation electrode,DNA will be drawn to the counterelectrode and concentrated near theopening of the separation channel. By floating the counterelectrode andinjecting using the separation electrode and the anode at the far end ofthe separation channels, concentrated product is electrokineticallyinjected.

C. INTEGRATION METHODS

The biochip also contains several different means for integrating thefunctional modules. These means involve the transport of liquids frompoint to point on the biochip, the control of flow rates for processesthat are flow-rate dependent, (e.g., some washing steps, particleseparation, and elution), the gating of fluid motion in time and spaceon the biochip (e.g., through the use of some form of valve), and themixing of fluids.

A variety of methods can be used for fluid transport and controlledfluid flow. One method is positive-displacement pumping, in that aplunger in contact with either the fluid or an interposing gas or fluiddrives the fluid a precise distance based on the volume displaced by theplunger during the motion. An example of such a method is a syringepump. Another method is the use of integrated elastomeric membranes thatare pneumatically, magnetically, or otherwise actuated. Singly, thesemembranes can be used as valves to contain fluids in a defined spaceand/or prevent premature mixing or delivery of fluids. When used inseries, however, these membranes can form a pump analogous to aperistaltic pump. By synchronized, sequential actuation of membranesfluid can be “pushed” from its trailing side as membranes on the leadingside are opened to receive the moving fluid (and to evacuate anydisplaced air in the channels of the device). A preferred method foractuation of these membranes is pneumatic actuation. In such devices,the biochip is comprised of fluidic layers, at least one of that hasmembranes, one side of that is exposed within the fluid channels andchambers of the device. The other side of the membrane is exposed to apneumatic manifold layer that is plumbed to a pressure source. Themembranes are opened or closed by the application of pressure or vacuum.Valves that are normally open or normally closed can be used, changingstate under the application of pressure or vacuum. Note that any gas canbe used for actuation, as the gas does not contact the fluids underanalysis.

Yet another method for driving fluids and controlling flow rates is toapply vacuum or pressure directly on the fluids themselves, by alteringthe pressure at the leading, trailing, or both menisci of the fluid.Appropriate pressures (typically in the range of 0.05-3 psig) areapplied. Flow rates also can be controlled by properly sizing thefluidic channels, as the flow rate is proportional to the pressuredifferential across the fluid and the hydraulic diameter to the fourthpower and inversely proportional to the length of the channel or theliquid plug and the viscosity.

Fluid gating can be achieved using a variety of active valves. Theformer can include piezoelectric valves or solenoid valves that can bedirectly incorporated into the chip, or applied to the biochip such thatports on the main chip body communicate with the valves, directing fluidinto the valves and then back into the chip. One drawback to these typesof valves is that for many applications, they are likely to be difficultto manufacture and too expensive to incorporate into disposableintegrated devices. A preferable approach is to use of membranes asvalves, as discussed above. For example, membranes actuated by 10 psigcan be used to successfully contain fluids undergoing PCR.

In some applications, capillary microvalves, which are passive valves,can be preferable. Essentially, microvalves are constrictions in theflow path. In microvalves, surface energy and/or geometric features suchas sharp edges can be used to impede flow when the pressure applied tothe fluid is below a critical valve, termed the burst pressure, which isgenerally given by the relation:

P_(valve)α(γ/d_(H))*sin(θ_(c))

where γ is the surface tension of the liquid, d_(H) is the hydraulicdiameter of the valve (defined as 4 * (cross-sectionalarea)/cross-sectional perimeter), and θ_(c) is the contact angle of theliquid with the valve surface.

Properties that make passive valves preferable for certain applicationsinclude: extremely low dead-volume (typically in the picoliter range),and small physical extent (each being only slightly larger than thechannels leading to and from the valve). Small physical extent allowsfor a high density of valves on a given surface of the biochip.Additionally, certain capillary valves are very simple to manufacture,consisting essentially of a small hole in a sheet of plastic, with orwithout a surface treatment. Judicious use of capillary valves canreduce the total number of membrane valves required, simplify theoverall manufacture and create a robust system.

Capillary valves implemented in devices of the invention are of twotypes: In-plane valves, in that the small channels and sharp corners ofthe valves are formed by creating “troughs” in one layer and bondingthis layer to a featureless lid (typically another layer of the device);and through-hole valves, in that small (typically 250 μm or less) holesare made in an intermediate layer between two fluidics-carrying layersof the device. In both cases, treatment with fluoropolymer can be usedto increase the contact angle of fluids in contact with the valves.

FIG. 7 shows the valving performance of these valves for liquids ofinterest, namely, deionized water and cycle sequencing reagent, as afunction of valve size for the case of fluoropolymer treatment. In bothcases, the expected dependence of valving pressure on valve dimension isobserved (Pressure ˜1/diameter). Through-hole valves have significantadvantages over in-plane valves. First, they are easier to manufacture,in that small through-holes can be readily made in a sheet of plastic,either by molding around posts, punching, die-cutting, drilling, orlaser-drilling after the valve layer has been created. In-plane valvesrequire fairly precise fabrication, and very fine valves (with highvalving pressures) necessitate the use of lithographic techniques tocreate the required molding or embossing tools. Second, through-holevalves can be more completely coated with fluoropolymer on “all sides.”The application of low-surface-tension fluoropolymer solution to a holeresults in complete coating of the internal walls of the hole bycapillary action. Coating of all sides of an in-plane valve requiresapplication of fluoropolymer to both the valve as well as the region ofthe mating layer that seals over the valve. As a result, typicalin-plane valves are formed without coating on the “roof” of the valve.

In machined prototypes, through-hole valves are both easier to implementand exhibit greater valving pressures, as illustrated in FIG. 7.

Mixing can be accomplished in a variety of ways. First, diffusion can beused to mix fluids by co-injecting the two fluids into a single channel,usually of small lateral dimension and of sufficient length such thatthe diffusion time

t _(D)=(width)²/(2*Diffusion constant)

is satisfied at the given flow rates. Unfortunately, this type of mixingis typically inadequate for mixing large volumes quickly, because thediffusion or mixing time scales with the channel width squared Mixingcan be enhanced in a variety of ways, such as lamination, in that thefluid stream is divided and recombined. (Campbell and Grzybowski Phil.Trans. R. Soc. Lond. A 2004, 362, 1069-1086); or through the use of finemicrostructure to create chaotic advection within the flow channel(Stroock et al., Anal. Chem. 2002. 74, 5306-4312). In systems usingactive pumps and valves, mixing can be accomplished by cycling fluidbetween two points on the device multiple times. Finally, the latteralso can be accomplished in systems using capillary valves. A capillaryvalve disposed between two channels or chambers acts as a pivot forfluid flow; as fluid flows from one channel into the other through thecapillary, the trailing meniscus is trapped if sufficiently low pressureis used to pump the fluid. Reversal of the pressure drives the fluidback into the first channel, and it is again pinned at the capillary.Multiple cycles can be used to efficiently mix components.

Approaches to separation and detection in microfluidic formats aredescribed in the U.S. patent application entitled “PLASTIC MICROFLUIDICSEPARATION AND DETECTION PLATFORMS”, and assigned U.S. Ser. No.12/080,745, filed on Apr. 4, 2008, which is incorporated by reference inits entirety (see, e.g., paragraphs 68-79, 94-98, therein).

The upper portion of FIG. 13 shows the construction of the integratedbiochip (1301) from two components which are bonded in or duringmanufacture. First, a 16-sample biochip (1302) combining the lysis,amplification, and sequencing features of the biochip of FIG. 1 with thesequencing product purification features of the biochip of FIG. 11 andsecond, a 16-lane plastic separation biochip (1303). Purified sequencingproduct can also be electrokinetically injected prior to separation.

D. FABRICATION METHODS

The devices of the invention can be primarily composed of plastics.Useful types of plastics include, but are not limited to: cyclic olefinpolymer (COP); cyclic olefin copolymer (COC); (both of that haveexcellent optical quality, low hygroscopicity, and high operatingtemperatures when of sufficient molecular weight); poly(methylmethacrylate) (PMMA) (readily machinable and can be obtained withexcellent optical properties); and polycarbonate (PC) (highly-moldablewith good impact resistance and a high operating temperature). Moreinformation about materials and fabrication methods are contained in theU.S. patent application entitled “METHODS FOR RAPID MULTIPLEXEDAMPLIFICATION OF TARGET NUCLEIC ACIDS” (U.S. Ser. No. 12/080,746) thathas been incorporated by reference (supra).

A variety of methods can be used to fabricate the individual parts ofthe biochip and to assemble them into a final device. Because thebiochip can be composed of one or more types of plastic, with thepossible inclusion of inserted components, the methods of interestpertain to creation of individual parts followed by post-processing ofparts and assembly.

Plastic components can be fabricated in several ways, includinginjection molding, hot embossing and machining. Injection molded partscan be comprised of both gross features (such as fluid reservoirs) aswell as fine features (such as capillary valves). In some cases, it canbe preferable to create fine features on one set of parts and largerfeatures on another set, because the approaches to injection molding ofthese differently-sized features can vary. For large reservoirs(measuring several (about 1-50 mm) mm on a side and with depths ofseveral mm (about 1-10 mm) and capable of accommodating 100 s of μL),conventional molding can be employed using machined injection moldingtools, or tools created by burning into a steel or other metal using agraphite electrode that has been machined to be a negative of the tool.

For fine features, both tool creation and molding process can be varied.Tools are typically created using a lithographic process on a substrateof interest (for example, isotropic etch of glass, or deep reactive ionetching or other processes on silicon). The substrate can then beelectroplated with nickel (usually after deposition of a chromium layerto promote adhesion) and the substrate removed, for example, by etchingin an acid. This nickel “daughter” plate is the injection molding tool.The molding process can be somewhat different than above, as well: Forfine, shallow features, compression-injection molding, in which the moldis physically compressed slightly after plastic has been injected intothe cavity, has been found to be superior to standard injection moldingin terms of fidelity, precision, and reproducibility.

For hot embossing, similar issues regarding gross and fine features asdiscussed above hold, and tools can be created as above. In hotembossing, plastic resin in the form of pellets, or as a pre-formedblank of material created through molding or embossing, can be appliedto the tool surface or a flat substrate. A second tool may then broughtinto contact at precisely controlled temperature and pressure in orderto raise the plastic above its glass transition temperature and to causematerial flow to fill the cavities of the tool(s). Embossing in a vacuumcan avoid the problem of air becoming trapped between tool and plastic.

Machining also can be employed to create parts. High-speed computernumerical controlled (CNC) machines can be used to create manyindividual parts per day from either molded, extruded, or solvent-castplastic. Proper choice of milling machine, operating parameters, andcutting tools can achieve high surface quality (surface roughnesses of50 nm are achievable in high-speed milling of COC (Bundgaard et al.,Proceedings of IMechE Part C: J. Mech. Eng. Sci. 2006, 220, 1625-1632).Milling can also be used to create geometries that can be difficult toachieve in molding or embossing and to readily mix feature sizes on asingle part (for example, large reservoirs and fine capillary valves canbe machined into the same substrate). Another advantage of milling overmolding or embossing is that no mold-release agents are needed torelease the fabricated part from a molding tool.

Post-processing of individual parts includes optical inspection (thatcan be automated), cleaning operations to remove defects such as burrsor hanging plastic, and surface treatment. If optical-quality surfacesare required in machined plastic, polishing with a vapor of a solventfor the plastic can be used. For example, for PMMA, dichloromethane canbe used, while for COC and COP, cyclohexane or toluene can be used.

Prior to assembly, surface treatments can be applied. Surface treatmentcan be performed to promote or reduce wetting (i.e., to change thehydrophilicty/hydrophobicity of the part); to inhibit the formation ofbubbles within microfluidic structures; to increase the valving pressureof capillary valves; and/or to inhibit protein adsorption to surfaces.Coatings that reduce wettability include fluoropolymers and/or moleculeswith fluorine moieties that are exposed to the fluid when the moleculesare adsorbed or bonded to the surfaces of the device. Coatings can beadsorbed or otherwise deposited, or they can be covalently linked to thesurface. The methods that can be used to make such coatings include dipcoating, passing coating reagent through the channels of the assembleddevice, inking, chemical vapor deposition, and inkjet deposition.Covalent bonds between coating molecules and the surface can be formedby treatment with oxygen or other plasma or UV-ozone to create anactivated surface, with either subsequent deposition or co-deposition ofthe surface treatment molecule on the surface (see, Lee et al.Electrophoresis 2005, 26, 1800-1806; and Cheng et al., Sensors andActuators B 2004, 99, 186-196.)

Assembly of component parts into the final device can be performed in avariety of ways. Inserted devices, such as filters, can be die-cut andthen placed with a pick-and-place machine.

Thermal diffusion bonding can be used, for example for the bonding oftwo or more layers of the same material, each of that is of uniformthickness. Generally, the parts can be stacked and the stack placed intoa hot press, where the temperature can be raised to the vicinity of theglass transition temperature of the material comprising the parts, tocause fusion at the interfaces between the parts. An advantage of thismethod is that the bonding is “general”, i.e., any two stacks of layersof roughly the same dimensions can be bonded, regardless of the internalstructure of the layers, because heat and pressure are applied uniformlyacross the layers.

Thermal diffusion bonding may also be used to bond more complex parts,such as those that are not planar on their bonding or opposing surfaces,by using specially-created bonding cradles. Such cradles conform to theouter surface of the layers to be bonded.

Other bonding variations include solvent-assisted thermal bonding, inthat a solvent such as methanol partially solubilizes the plasticsurface, enhancing bond strength at a lower bonding temperature. Afurther variation is the use of spin-coated layers of lower-molecularweight material. For example, a polymer of the same chemical structurebut of a lower molecular weight than the substrate components can bespun onto at least one layer to be bonded, the components assembled, andthe resulting stack bonded, by diffusion bonding. During thermaldiffusion bonding, the low-molecular weight components can pass throughtheir glass transition temperature at a lower temperature than thecomponents and diffuse into the substrate plastic.

Adhesives and epoxies can be used to bond dissimilar materials and arelikely to be used when bonding components fabricated in different ways.Adhesive films can be die cut and placed on components. Liquid adhesivemay also be applied through spin-coating. Inking of adhesive ontostructured parts (such as in nanocontact printing) can be successfullyused to apply adhesive to structured surfaces without a need to “direct”the adhesive onto particular areas.

In one example, a biochip of the invention can be assembled as shown inFIG. 6. Layers 1 and 2 can be aligned by included features (e.g., pinsand sockets); separately, layers 3 and 4 can be similarly aligned byincluded features. The layer 1 plus layer 2 stack can be inverted andapplied to the layer 3 plus layer 4 stack and then entire stack can bebonded.

E. EXAMPLES Example 1 Integrated Biochip for Nucleic Acid Extraction andAmplification

An integrated biochip for DNA extraction and amplification by PCR isshown in FIG. 1. This 4-sample device integrates the functions ofreagent distribution and metering; mixing of reagents with samples;delivery of samples to a thermal cycling portion of the chip; andthermal cycling. The same biochip is used in Example 2 below and hasadditional structures for performance of cycle sequencing.

The biochip was constructed of 4 layers of thermoplastic as shown inFIGS. 2-5. The 4 layers are machined PMMA and have thicknesses of thelayers are 0.76 mm, 1.9 mm, 0.38 mm, and 0.76 mm, respectively, and thelateral size of the biochip was 124 mm×60 mm. In general, biochips of atthree or more layers allow the use of an indefinite number of commonreagents to be divided among multiple assays: two fluidic layers and onelayer that at least contains through-holes, enabling fluidic channels inthe outer layers to ‘cross-over’ one another. (It will be recognizedthat special cases exist—such as the use of only one common reagentamong multiple samples—that do not necessitate a three-layerconstruction.) The choice of 4 layers was made for compatibility withconstruction of chips for other functions (such as ultrafiltration,Example 3) and full integration (Example 4).

The channels of the biochip were of cross-sectional dimensions rangingfrom 127 μm×127 μm to 400 μm×400 μm, while reservoirs ranged from 400μm×400 μm in cross-section to 1.9×1.6 mm; both channels and reservoirsextend for distances as short as 0.5 mm to several 10s of mm. Thecapillary valves used in the biochip were of 127 μm×127 μm size for“in-plane” valves and 100 μm in diameter for through-hole capillaryvalves.

Certain channels, reservoirs, and capillary valves of the four machinedlayers were treated with a hydrophobic/olephobic material, PFC 502A(Cytonix, Beltsville, Md.). Surface treatment was performed by coatingwith a wetted Q-tip followed by air-drying at room temperature. Thedried fluoropolymer layer was less than 10 μm thick as determined byoptical microscopy. Surface treatment serves two purposes: to preventthe formation of bubbles within liquids, especially withinlow-surface-tension liquids, such as cycle sequencing reagent, which canoccur as the liquid rapidly wets the walls of channels or chambers (and“closes off” a bubble before the air can be displaced), and to enhancethe capillary burst pressure at that capillary valves resist liquidflow. The regions left untreated were the thermal cycling chambers forPCR and cycle sequencing.

After surface treatment, the layers were bonded as shown in FIG. 6.Bonding was performed using thermal diffusive bonding, in that the stackof components was heated under pressure to a temperature near the glasstransition temperature (T_(g)) of the plastic. A force of 45 lbs wasapplied over the entire 11.5 square inch biochip for 15 minutes during athermal bonding profile consisting of a ramp from ambient temp to 130°C. in 7.5 minutes, a hold at 130° C. for 7.5 minutes, and rapid coolingto room temperature.

Pneumatic instrumentation was developed for driving fluids within thebiochips of the invention. Two small peristaltic pumps provided pressureand vacuum. Positive pressure output was divided among three regulatorsthat have the range of approximately 0.05-3 psig. The vacuum was portedto a regulator with an output vacuum of approximately (−0.1)-(−3) psig.A fourth, higher pressure was taken from a cylinder of N₂ to a furtherregulator or alternatively from a higher-capacity pump. The positive andnegative pressures were applied to a series of 8 pressure-selectormodules. Each module was equipped with solenoid valves that could choosean output pressure to be transmitted to the biochip from among the 5inputs. The output pressure lines terminated on at least one pneumaticinterface. This interface clamped to the chip with O-rings positionedover the chip ports on the input side of the chip (the ports along thetop of the figures).

Immediately above the biochip ports were additional solenoid valves(i.e., gate valves; 8 per interface) that accept the output pressurelines from the pressure-selector modules. These valves, in closeproximity, to the chip provide a low dead-volume interface(approximately 13 μL) between the pressure line and the chip. A lowdead-volume interface can prevent unintentional motion of certainliquids on the biochip when pressure is applied to move other liquids(the small gas volume between the liquid plug and the closed valvedetermines the maximum amount the plug can move, for example, due tocompression of the gas as pressure is applied). All pressure-selectorvalves and gate valves were operated under computer control using ascript-based LabView™ program. An important feature of this system isthat short pressure cycles times are possible. Some fluidic controlevents could be performed that required pulses of pressure as short as30 msec and/or complex pressure profiles could be utilized wherepressure could be switched from one value to another (i.e., oneregulator to another) rapidly (that is, with time lags of no more than10-20 msec).

The samples consisted of a bacterial suspension of approximately 10⁶cells/mL of E. coli DH5 transformed with pGEM sequencing plasmid insert(pUC18 sequencing target). PCR reagent consisted of dNTPs KOD TaqPolymerase (Novagen, Madison, Wis.) at concentration 0.1 μM

A 1.23 μL sample of the bacterial suspension was added to each of thefour ports 104, each comprising through holes 202 and 336 in layers 1and 2, respectively. The sample then resided in sample channels 303 inlayer 2. Next, 10 μL of PCR reagent was added to port 105 comprising ofthrough holes 217 and 306 in layers 1 and 2. The PCR reagent thenresided in chamber 307 in layer 2 (see, FIG. 8a ). A port for theevacuation of displaced air for the PCR reagent was port 107, comprising109 and through-holes 203+305.

In operation, air displaced by samples and downstream processes (such asmetering of reagents, mixing of fluids) was evacuated through ports onthe output end of the chip, 108, comprised of through-holes 227. Thefinal volume of the PCR reaction can be increased or decreased asdesired.

The biochip was placed in the pneumatic manifold described above. Thefollowing automated pressure profile was performed with no delaysbetween steps. Unless otherwise noted, the pneumatic interface valves,corresponding to ports along the input side of the chip, were closedduring all steps.

A pressure of 0.12 psig was applied to ports 104 for 15 sec to drive thesamples down channels 303 to through-hole 304. The samples passedthrough through-hole 304 and emerged on the other side of layer 2 insample chamber 204 of layer 1 and were driven to the first mixingjunction 205. At the first mixing junction the samples were retained bycapillary valves 210 (see, FIGS. 8b-c ).

A pressure of 0.12 psig was applied to port 105 for 10 sec to drive thePCR reagent through through-hole 320. The PCR reagent emerged on theother side of layer 2 in distribution channel 208, and moved into themetering chambers 209, which define a volume of reagent equal to thesample volume, where they were retained by capillary valves 211 atmixing junction 205. (see, FIG. 8d ).

A pressure of 0.12 psig was applied to port 107 (comprised ofthrough-holes 203 and 305) with port 105 open to atmosphere for 3 sec toempty channel 208 (see, FIG. 8e ).

A pressure of 0.8 psig was applied to ports 107 and 105 for 0.03 sec anda pressure of 0.7 psig was simultaneously applied to ports 104 for 0.03sec to initiate mixing of the samples and PCR reagents by burstingliquids past the capillary valves 210 and 211 (see, FIG. 8f ).

A pressure of 0.12 psig was applied to ports 104 and 107 for 10 sec topump the samples and PCR reagents into mixing channels 214, withretention at capillary valves 210 and 211. Passage through the mixingbulbs 212 into the constrictions 213 created added hydraulic resistanceto flow, decreasing the high velocity imparted by the previous highpressure pulse.

A pressure of 0.7 psig was applied to ports 104 and 107 for 0.03 sec todetach the liquid from capillary valves 210 and 211 (see FIG. 8g ).

A pressure of 0.12 psig was applied for 3 sec to ports 104 and 107 topump liquid through mixing channel 214 to capillary valves 219, wherethey were retained (see, FIG. 8h ).

A pressure of 0.7 was psig applied for 0.1 sec to ports 104 and 107 todrive the mixture of the samples and PCR reagents through through-holes315 and 402 and through the body of layers 2 and 3, and into PCR chamber502 (see, FIG. 8i ).

A pressure of 0.12 psig was applied for 3 sec to ports 104 and 107 tocomplete pumping of the mixture of the samples and PCR reagents intochamber 502. The leading edge of the mixture of the samples and PCRreagents then passed through through-holes 403 and 316, emerged intolayer 1, and was pinned at capillary valve 220 (see, FIG. 8j ).

The biochip was then pressurized to 30 psig N₂ and thermally cycled forPCR amplification via a Peltier using a gas bladder compressionmechanism as described the U.S. patent application entitled, “METHODSFOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS”, U.S. Ser.No. 12/080,746, filed on Apr. 4, 2008; and in International PatentApplication Serial No. PCT/US08/53234, Attorney Docket No. 07-084-WO,filed 6 Feb. 2008 and entitled, “DEVICES AND METHODS FOR THE PERFORMANCEOF MINIATURIZED IN VITRO ASSAYS,” each of which are hereby incorporatedby reference in their entirety.

Sample, reagent volumes, and PCR chamber sizes were chosen such that theliquid filled the region between valves 219 and valves 220. As a resultthere liquid/vapor interfaces of small cross sectional area (typically127 μm×127 μm) were located approximately 3 mm from the thermally cycledbottom surface of layer 4. The application of pressure during thermalcycling inhibited outgassing by dissolved oxygen in the sample. Thesmall cross-sectional area of the liquid/vapor interface and distancefrom the Peltier surface both inhibited evaporation.

The observed temperature at the top of the biochip during cycling neverexceeded 60° C., and, as a result, the vapor pressure at theliquid/vapor interfaces was significantly lower than it would have beenfor such interfaces if they were within the PCR chamber. For a 2 μLsample, 1.4 μL of which is within chamber 502 and the remainder iswithin the through-holes and capillary valves, the observed evaporationwas less than 0.2 μL over 40 cycles of PCR. The volume of non-cycledfluid—0.6 μL in this case—can be reduced by the choice of smallerdiameters for the through-holes.

PCR was performed using the following temperature profile:

-   -   Heat lysis of bacteria for 3 min at 98° C.    -   40 cycles of the following        -   Denaturation at 98° C. for 5 sec        -   Annealing at 65° C. for 15 sec        -   Extension at 72° C. for 4 sec        -   Final extension at 72° C./2 min            The PCR product was retrieved by flushing the chamber 502            with ˜5 μL deionized water and analyzed by slab gel            electrophoresis. PCR yield was up to 40 ng per reaction,            much more than required for subsequent sequencing reactions.            In this application, bacterial nucleic acids were generated            merely by lysing bacteria. Nucleic acids can be subjected to            purification as required, a process that can improve the            efficiency of amplification, sequencing, and other            reactions.

Example 2

Integrated Biochip for Distribution of Cycle Sequencing Reagent, Mixingwith PCR Product, and Cycle Sequencing

The biochips described in Example 1 were used. PCR product generated intubes using the protocol outlined in Example 1 was added to both sampleand PCR reagent ports of the biochip as described above. 50 μL of acycle sequencing reagent (BigDye™ 3.1/BDX64, MCLab, San Francisco) wasadded to port 106 (comprised of through-holes 215 and 308) and chamber309. After installation of two pneumatic interfaces (one for the inputand one for the output end of the chip), the PCR product was processedas described in Example 1 through to the PCR chamber, but without thePCR thermal cycling step. The disposition of the fluids in the chip wasas shown in FIG. 9 a.

The following pressure profile was carried out using the pneumaticsystem software; all solenoid valves corresponding to chip ports wereclosed unless otherwise noted:

1. A pressure of 0.1 psig was applied to port 106 with ports 109 open toatmosphere for 10 sec to pump cycle sequencing reagent into channel 310(see, FIG. 9b ).

2. A pressure of 0.7 psig was applied for 0.2 sec on ports 106 and 108(comprised of through-holes 216 and 314) to drive cycle the sequencingreagent from channel 304 through through-holes 311, through the body oflayer 2, and into in the cycle sequencing reagent metering chambers 218on layer 1 (see, FIG. 9c ).

3. A pressure of 0.1 psig was applied to port 106 with ports 109 open toatmosphere, driving the cycle sequencing reagent to the capillary valves221, where it was retained (see, FIG. 9d ).

4. A pressure of 0.1 psig was applied to port 108 with port 106 open toatmosphere for 1 sec to drive excess cycle sequencing reagent backwardsinto chamber 101, leaving channel 310 empty (see, FIG. 9e ).

5. A pressure of 0.7 psig was applied to ports 104 and 107 for 0.1 secwith ports 109 open to atmosphere to drive PCR product past capillaryvalve 220 and into through hole 317, passing through the body of layer 2and through-hole 404 in layer 3, and into the cycle sequencing chamber503 of layer 4 (see, FIG. 9f ).

6. A pressure of 0.1 psig was applied to ports 109 with ports 104 and107 open to atmosphere for 5 sec to drive PCR sample back to thethrough-holes. Capillary action retained the liquids at the entrance ofthe through-hole, preventing a trapped air bubble from appearing betweenthe PCR product and chamber 503 (see, FIG. 9g ).

7. A pressure of 0.7 psig was applied to port 108 for 0.2 sec with ports109 open to atmosphere to drive cycle sequencing reagent into chamber503, while simultaneously applying 0.1 psig to ports 104 and 107, tocontact the PCR product with the sequencing reagent (see, FIG. 9h ).

8. A pressure of 0.1 psig was applied for 10 sec to ports 104, 107 and108 with ports 109 open to atmosphere to drive PCR product and Sangerreagent into the chamber. The trailing meniscus of the PCR product andthat of the sequencing reagent were pinned at the capillary valves 220and 221 (see, FIG. 9i ).

9. 5 vacuum pulses of 0.25 psig vacuum and duration 0.1 sec were appliedto port 108 with ports 109 open to atmosphere to draw both liquidspartially backwards into reagent metering chamber 218 (see, FIG. 9j ).

10. A pressure of 0.1 psig was applied to ports 104, 107, and 108 withports 109 open to atmosphere for 10 sec to pump the mixture back intochamber 503, with the trailing meniscus being pinned at capillary valvesas in step 8. (see, FIG. 9k ).

Steps 9-10 were repeated an additional two times to effect mixing of thesequencing reagent and PCR product.

The biochip was then pressurized to 30 psig N₂ and thermally cycledusing the following temperature profile:

-   -   95° C./1 min initial denaturation        -   30 cycles of the following        -   Denaturation at 95° C. for 5 sec        -   Annealing at 50° C. for 10 sec        -   Extension at 60° C. for 1 min            Samples (see, FIG. 9l ) were retrieved and purified by            ethanol precipitation and analyzed by electrophoretic            separation and laser-induced fluorescence detection on the            Genebench™ instrument as described infra (Part II, Example            5). Phred quality analysis yielded 408+/−57 QV20 bases per            sample.

Example 3 Ultrafiltration in 4-Sample Biochips

A 4-sample biochip for the performance of sequencing productpurification was constructed of four layers, as discussed in Example 1,and is shown in FIG. 11. One additional element in construction was theultra-filtration (UF) filter 1116, which is cut to size and placedbetween layers 3 and 4 prior to thermal bonding. The creation of a goodbond around the UF filter necessitated the use of layer 3. Layers 3 and4 create uninterrupted perimeters around the filter, because allchannels leading to and from the filter are in the bottom of layer 2.(Bonding directly between layer 2 and 4, for example, leaves a poor bondto the filter where channels cross the filter.) In this example, aregenerated cellulose (RC) filter of molecular weight cut-off (MWCO) 30kD was used (Sartorius, Goettingen, Germany). A variety of other MWCOs(10 kD, 50 kD, and 100 kD) have been examined, as has an alternativematerial, polyethersulfone (Pall Corporation, East Hills, N.Y.).

1. Four 10 μL samples of cycle sequencing product generated in tubereactions using pUC18 template and KOD enzyme were added to ports 1104in the first layer and driven through channel 1105 in the second layerto the chamber 1106 in the second layer. 200 μL of deionized water wasadded to port 1120 (a through-hole in the first layer) to reservoir 1121in the second layer. The biochip was then installed in two pneumaticinterfaces.

The following pressure profile was carried out using the pneumaticsystem software. All solenoid valves corresponding to biochip ports wereclosed unless otherwise noted.

2. A pressure of 0.09 psig was applied to ports 1104 with ports 1119open to atmosphere for 5 sec to drive the sequencing product tocapillary valves 1108 in layer 1, where they were retained.

3. A pressure of 0.6 psig was applied to ports 1104 with ports 1119 opento atmosphere for 0.1 sec to burst the samples through capillary valves1108 in layer 1 and deliver them through through-holes 1111 in layer 2into UF input chambers 1112 in layer 2.

4. A pressure of 0.09 psig was applied for to ports 1104 with ports 1119open to atmosphere for 10-30 sec (different times were used in differentexperiments) to complete delivery of sequencing product to chambers1112. Sequencing product was retained by capillary valves 1113 in layer2 (see FIGS. 12a and 12b ).

5. A pressure of 0.8 psig was applied to port 1124 with ports 1119 and1104 open to atmosphere for 0.5 sec to drive sequencing product throughvalves 113 into filtration chambers 1115. This also cleared inputcapillary valves 1108 of retained liquid.

6. A pressure of 0.09 psig was applied to port 1124 with ports 1119 opento atmosphere for 10-30 sec to complete delivery of the sequencingproduct to chamber 1115. Sequencing product was retained at valve 1113(see, FIG. 12c ).

7. A pressure of 7.5 psig was slowly applied to all ports of the chipfor ultrafiltration. During ultrafiltration, the sequencing productmeniscus remains pinned at 1113 while the leading edge of the liquid“retracts” as liquid was driven through the filter 1116. 10 μL ofsequencing product required ˜120 sec for filtration. The pressure wasreleased after filtration (see, FIGS. 12c and 12d ).

8. A pressure of 0.09 psig was applied to port 1120 with port 1124 opento atmosphere for 3 sec to drive water into channel 1122 (in layer 4)and partially-fill overflow chamber 1123 (see, FIG. 12e ).

9. A pressure of 0.8 psig was applied to ports 1120 and 1124 with ports1119 open to atmosphere to drive water through through-hole capillaryvalves 1110 in channel 1122 into chambers 1112.

10. A pressure of 0.09 psig was applied to port 1120 with ports 1119open to atmosphere for 10-30 sec to complete delivery of liquid tochambers 1112, where it was retained by valves 1113. (see, FIG. 12f ).

11. A pressure of 0.09 psig was applied to port 1124 with port 1120 opento drive water in chamber 1123 and channel 1122 back into chamber 1121(see, FIG. 12g ).

12. A pressure of 0.8 psig was applied to port 1124 with ports 1119 and1104 open to atmosphere for 0.5 sec to drive water through valves 113into filtration chambers 1115. This also cleared input capillary valves1108 of retained liquid.

13. A pressure of 0.09 psig was applied to port 1124 with ports 1119open to atmosphere for 10-30 sec to complete delivery of water tochamber 1115. Sequencing product was retained at valve 1113 (see, FIG.12h ).

The water was driven through the UF filter as in step 6 above,completing the first wash Steps 8-13 were repeated one additional time.

Steps 8-12 were repeated to partially-fill chambers 1115 with a finalvolume of water used for elution (see, FIG. 12k ).

Vacuum of 1.6 psig was applied to ports 1104 with all other ports closedfor 1 sec, drawing some water from chambers 1115 into chambers 1112 (themaximum motion being dictated by the creation of a vacuum of equalmagnitude in the dead-space between the meniscus of the liquid and thesolenoid valves corresponding to ports 1119), (see, FIG. 12l ).

Ports 1104 were opened to atmosphere for 1 sec, allowing the liquid tomove back into chamber 1115 due to the partial vacuum generated betweenthe liquid and the valves corresponding to ports 1119 (see, FIG. 12m ).

16-17 was repeated 50× to create 50 elution cycles.

A pressure of 0.09 psig was applied to port 1124 for 10 sec with ports1119 open to atmosphere to drive liquids such that its trailing meniscuswas pinned at 1113.

A pressure of 0.7 psig/0.05sec was applied to port 1124 with ports 1119open to atmosphere to detach the eluent (see, FIG. 12n ).

The samples were retrieved and run directly on Genebench™ as described,yielding up to 479 QV20 bases.

Example 4 Fully Integrated Biochip for Nucleic Acid Extraction, TemplateAmplification, Cycle Sequencing, Purification of Sequencing Product, andElectrophoretic Separation and Detection of Purified Product

FIG. 13 illustrates an embodiment of a 16-sample biochip, 1301, whichcombines the lysis and extraction, template amplification, and cyclesequencing functions of the biochip of FIG. 1; the ultrafiltrationfunction of the chip of FIG. 11; and electrophoretic separation anddetection. The process through ultrafiltration is carried out bysub-component 1302 and can be performed as described in examples 1, 2,and 3; transfer points 1304 on the bottom surface of 1302 are alignedwith input wells 1305 on the separation sub-component 1303.

Injection is performed electrokinetically with a pre-concentration stepusing counterelectrodes. The input well 1305, illustrated in FIG. 14,consists of a liquid receiving well 1401; a main separation electrode,1402; and a counterelectrode 1403. Separation channel 1306 opens intothe bottom of well reservoir 1401. The separation electrode is typicallyplatinum or gold coated, and is preferably a planar gold-coatedelectrode that substantially covers 1, 3, or 4 of the internal surfacesof 1401. The counterelectrode is a thin gold, steel, or platinum wire(typically 0.25 mm in diameter) that has been coated with a thin layer(˜10 μm) of cross-linked polyacrylamide. This forms a hydrogelprotection layer on the electrode. In panel d, purified sequencingproduct (black dots within 1401) have been transferred to the well.Applying positive potential between 1402 and 1403, negatively chargedsequencing product is drawn toward 1403, as in panels c-d. The hydrogellayer on 1403 prevents sequencing product from contacting the metalelectrode and thus prevents electrochemistry and damage of thesequencing product. The counterelectrode 1403 is then allowed to floatwith respect to 1402. A positive potential is then applied between mainseparation electrode 1402 and the anode (not shown) at the far end ofseparation channel 1306. This allows product to be injected (panel e)and to electrophoresis down 1306 for separation and detection (panel f).As illustrated in FIG. 14, this scheme allows the concentration ofsequencing product in the vicinity of the end of channel 1306 to beincreased significantly relative to the concentration with that it isdelivered from ultrafiltration. While such concentration is desirablefor some applications, it is not necessary in all cases. In such cases,the well of FIG. 14 without the counterelectrode 1403 can be used toperform EKI directly. Alternatively, the single electrode in the loadingwell may be one half of a cross-T or double-T injector (see, forexample, the U.S. patent application entitled, “PLASTIC MICROFLUIDICSEPARATION AND DETECTION PLATFORMS”, U.S. Ser. No. 12/080,745, filed onApr. 4, 2008).

Separation occurs in separation channels 1306, and detection occurs vialaser-induced fluorescence in the detection region 1307. In thisbiochip, a recess 1308 is provided to allow, for example, a Peltierblock (not shown) to mate with the lower surface of 1301 to providethermal cycling for PCR and cycle sequencing. Pneumatic interfaces (notshown) within the instrument clamp to the ends of the chip to providemicrofluidic control.

II. SEPARATION AND DETECTION SYSTEMS A. Detailed Description ofSeparation And Detection Components and Their Uses

1. Separation Instrument

DNA separation is carried out on a biochip and instrumentation asdescribed in U.S. Patent Application Publication No. US2006-0260941-A1.Separation chips can be glass (see, U.S. Patent Application PublicationNo. US2006-0260941-A1) or plastic (the U.S. patent application entitled,“PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS”, U.S. Ser. No.12/080,745, filed on Apr. 4, 2008), each of which are herebyincorporated by reference in their entirety.

2. Excitation and Detection Instrumentation

The instrument comprises excitation and detection subsystems forinteracting with and interrogating a sample. Samples typically includeone or more biological molecules (including but not limited to DNA, RNA,and proteins) that are labeled with dyes (e.g., fluorescent dyes). Theexcitation subsystem comprises an excitation source or sources and anexcitation beam path with optical elements including lenses, pinholes,mirrors and objectives, to condition and focus the excitation source inan excitation/detection window. Optical excitation of a sample can beaccomplished by a series of laser types, with emission wavelengths inthe visible region, between 400 to 650 nm. Solid state lasers canprovide emission wavelengths of approximately 460 nm, 488 nm, and 532nm. These lasers include, for example, the Compass, Sapphire and Verdiproducts from Coherent (Santa Clara, Calif.). Gas lasers includeargon-ion and helium neon with emission in the visible wavelengths atapproximately 488 nm, 514 nm, 543 nm, 595 nm, and 632 nm. Other laserswith emission wavelengths in the visible region are available fromCrystaLaser (Reno, Nev.). In one embodiment, a 488 nm solid state laserSapphire 488-200 (Coherent, Santa Clara, Calif.) can be utilized. Inanother embodiment, a light source with wavelength beyond the visiblerange can be used for exciting dyes having absorption and/or emissionspectra beyond the visible range (e.g., infrared or ultra-violetemitting dyes). Alternatively optical excitation can be achieved by theuse of non-laser light sources with emission wavelengths appropriate fordye excitation, including light emitting diodes, and lamps.

The detection subsystem comprises one or more optical detectors, awavelength dispersion device (which performs wavelength separation), andone or a series of optical elements including, but not limited to,lenses, pinholes, mirrors and objectives to collect emitted fluorescencefrom fluorophore-labeled DNA fragments that are present at theexcitation/detection window. The fluorescence emitted can be from asingle dye or a combination of dyes. In order to discriminate the signalto determine its contribution from the emitting dye, wavelengthseparation of the fluorescence can be utilized. This can be achieved bythe use of dichroic mirrors and bandpass filter elements (available fromnumerous vendors including Chroma, Rockingham, Vt.; and Omega Optical,Brattleboro, Vt.). In this configuration, the emitted fluorescencepasses through a series of dichroic mirrors where one portion of thewavelength will be reflected by the mirror to continue traveling downthe path, and the other portion will pass through. A series of discretephotodetectors, each one positioned at the end of the dichroic mirrorwill detect light over a specific range of wavelengths. A bandpassfilter can be positioned between the dichroic mirror and photodetectorto further narrow the wavelength range prior to detection. Opticaldetectors that can be utilized to detect the wavelength-separatedsignals include photodiodes, avalanche photodiodes, photomultipliermodules, and CCD cameras. These optical detectors are available fromsuppliers such as Hamamatsu (Bridgewater, N.J.).

In one embodiment, wavelength components are separated by the use ofdichroic mirrors and bandpass filters and these wavelength componentsare detected with Photomultiplier Tube (PMT) detectors (H7732-10,Hamamatsu). The dichroic mirror and bandpass components can be selectedsuch that incident light on each of the PMTs consists of a narrowwavelength band corresponding to the emission wavelength of thefluorescent dye. The band pass is typically selected to be centeredabout the fluorescent emission peak with a band pass of wavelength rangeof between 1 and 50 nm. The system is capable of eight color detectionand can be designed with eight PMTs and a corresponding set of dichroicmirrors and bandpass filters to divide the emitted fluorescence intoeight distinct colors. More than eight dyes can be detected by applyingadditional dichroic mirrors, bandpass filters and PMT. FIG. 15 shows thebeam path for discrete bandpass filter and dichroic filterimplementation. An integrated version of this wavelength discriminationand detection configuration is the H9797R, Hamamatsu, Bridgewater, N.J.

Another method of discriminating the dyes that make up the fluorescencesignal involves the use of wavelength dispersive elements and systemssuch as prisms, diffraction gratings, transmission gratings (availablefrom numerous vendors including ThorLabs, Newton, N.J.; and Newport,Irvine, Calif.; and spectrographs (available from numerous vendorsincluding Horiba Jobin-Yvon, Edison, N.J.). In this mode of operation,the wavelength components of the fluorescence are dispersed over aphysical space. Detector elements placed along this physical spacedetect light and allow the correlation of the physical location of thedetector element with the wavelength. Detectors suitable for thisfunction are array-based and include multi-element photodiodes, CCDcameras, back-side thinned CCD cameras, multi-anode PMT. One skilled inthe art will be able to apply a combination of wavelength dispersionelements and optical detector elements to yield a system that is capableof discriminating wavelengths from the dyes used in the system.

In another embodiment, a spectrograph is used in place of the dichroicand bandpass filters to separate the wavelength components from theexcited fluorescence. Details on spectrograph design is available inJohn James, Spectrograph Design Fundamental, Cambridge, UK: CambridgeUniversity Press, 2007. The spectrograph P/N MF-34 with a concaveholographic grating with a spectral range of 505-670 nm (P/N 532.00.570)(HORIBA Jobin Yvon Inc, Edison, N.J.) is used in this application.Detection can be accomplished with a linear 32-element PMT detectorarray (H7260-20, Hamamatsu, Bridgewater, N.J.). Collected fluorescenceis imaged on the pinhole, reflected, dispersed, and imaged by theconcave holographic grating onto the linear PMT detector that is mountedat the output port of the spectrograph. The use of a PMT-based detectortakes advantage of the low dark noise, high sensitivity, high dynamicrange, and rapid response characteristic of PMT detectors. The use of aspectrograph and multi-element PMT detector for detection of excitedfluorescence allows for flexibility in the number of dyes and theemission wavelength of dyes that can be applied within the systems andwithin the lane, without the need for physically reconfiguring thedetection system (dichroic, bandpass and detectors) of the instrument.The data collected from this configuration is a wavelength dependentspectra across the visible wavelength range for each scan for each lane.Generating a full spectrum per scan provides dye flexibility both interms of dye emission wavelength and number of dyes that can be presentwithin a sample. In addition, the use of the spectrometer and linearmulti-element PMT detector also allows for extremely fast read-out ratesas all the PMT elements in the array are read-out in parallel. FIG. 16shows the beam path for multi-element PMT and spectrographimplementation.

Instruments may employ a staring mode of operation, to detect multiplelanes simultaneously and multiple wavelengths simultaneously. In oneconfiguration, the excitation beam is simultaneously impinged on alllanes at the same time. The fluorescence from this is collected by a twodimensional detector such as a CCD camera or array. In this staring modeof collection, a wavelength dispersive element is used. One dimension ofthe detector represents the physical wavelength separation, while theother dimension represents the spatial or lane-lane separation.

For simultaneous excitation and detection of multiple samples, ascanning mirror system (62) (P/N 6240HA, 67124-H-0 and 6M2420X40S100S1,Cambridge technology, Cambridge Mass.) is utilized to steer both theexcitation and detection beam paths in order to image each of the lanesof the biochip. In this mode of operation, the scanning mirror steersthe beam paths, scanning sequentially from lane to lane from the firstlane to the last lane, and the repeating the process again from thefirst lane to the last lane again. A lane-finding algorithm such as thatdescribed in U.S. Patent Application Publication No. US2006-0260941-A1is used to identify location of lane.

An embodiment of an optical detection system for simultaneous multiplelane and multi dye detection is shown in FIG. 16. The fluorescenceexcitation and detection system 40 excites the components separated byelectrophoresis of a DNA sample (e.g., containing DNA fragmentsfollowing amplification of a set of STR loci) by scanning an energysource (e.g. a laser beam) through a portion of each of themicrochannels while collecting and transmitting the induced fluorescencefrom the dye to one or more light detectors for recordation, andultimately analysis.

In one embodiment, the fluorescence excitation and detection assembly 40includes a laser 60, a scanner 62, one or more light detectors 64, andvarious mirrors 68, spectrograph, and lenses 72 for transmitting a laserbeam emitted from the laser 60 through opening 42 to the test module 55and back to the light detectors 64. The scanner 62 moves the incominglaser beam to various scanning positions relative to the test module 55.Specifically, the scanner 62 moves the laser beam to a pertinent portionof each micro channel within the test module 55 to detect respectiveseparate components. The multi element PMT 64 collects data (e.g. thefluorescent signals from DNA fragments of varying length) from the testmodule 55 and provides the data electronically through a cable attachedto a port to a data acquisition and storage system located outside theprotective cover. In one embodiment, the data acquisition and storagesystem can include a ruggedized computer available from OptionIndustrial Computers (13 audreuil-Dorion, Quebec, Canada).

In another embodiment (a “staring mode”), the excitation source isincident on all the detection spots simultaneously, and fluorescencefrom all detection spots is collected simultaneously. Simultaneousspectral dispersion (wavelength spectra of detected fluorescence) andspatial dispersion (detection spots) can be performed with a twodimensional detector array. In this configuration, the 2-dimensionaldetector array is positioned in the system such that spectral componentsare imaged and detected across one dimension of the array detector(row), while spatial components are imaged and detected across the otherdimension of the array detector.

A preferred instrument utilizes a scanning mode of operation, ratherthan a “staring” mode. In scanning mode, signal for each channel isrequired to be collected, integrated, and read-out while the scanner iscoincident with the lane being interrogated and before it is incident onthe next channel. A detector with fast readout allows for optimal lightcollection and integration, translating into higher signal to noiseperformance. Ideally, the read-out time of the detector should besignificantly less than the total time which the scanner is coincidentwith the channel. The multi-element PMT can be read-out in less than 0.7ms and this read-out time is far less than the integration time fordetection for each individual channel.

Fluorescence incident on the pinhole can be dispersed by the gratingaccording to its wavelength composition and focused onto the linearmulti-anode PMT detector array. The detector provides 32 currentoutputs, one for each of the elements in the array that correspond tothe number of photons incident on the element. During multiple samples(or lanes) detection, when the laser is in position exciting theselected lane, the integrator circuitry will integrate the PMT outputcurrent to generate an output voltage proportional to the integrated PMTcurrent. At the same time, the single ended output voltage is convertedto differential mode using the Analog Devices (Norwood, Mass.)differential driver IC (P/N SSM2142). At the end of the integration time(defined by scan rate and number of lane), the data acquisition systemwill read the differential signal and save the data in its buffer. Afterthe data have been saved, the data acquisition system will move thescanner to shift the laser beam to the next selected lane, at the sametime resetting the integrator circuitry.

Each single element PMT module has its own integrator circuitry. For an8 color detection system, there are 8 PMT modules and 8 integratorcircuitries. Additional colors can be added using corresponding numbersof PMT modules and integrator circuitry.

Since each of the PMT elements (H77260-20, Hamamatsu, Japan) has asimilar or more rapid signal response as a single PMT tube (H7732-10,Hamamatsu, Japan), and the readout is in parallel, this detector is ableto operate very rapidly. When coupled with the spectrometer, thisspectrometer and multi-anode detector system is able to provide fullspectral scans across the visible spectrum (450 nm to 650 nm) withreadout-times of less than 0.1 ms.

The ability to provide fast refresh rates allows thisspectrometer/detector system to be applied to scanning modeimplementation of detection of multiple lanes sequentially within asingle run. The use of PMT based detectors provides for low noise, highsensitivity and high dynamic range, and fast response. The 140 mmspectrometer with a concave holographic grating (Horiba Jobin-Yvon) andmultianode PMT detector is the H7260-20 detector (Hamamatsu, Japan).Other spectrometers configurations and multi-anode PMT detectors canalso be used for this application.

Determination of nucleotide bases from the electrophoregrams wasachieved using signal processing algorithms to correct, filter, andanalyze the data. This process consisted of locating a callable signal,correcting the signal baseline, filtering out noise, removing colorcross-talk, identifying signal peaks, and determining associated bases.Locating the callable signal was performed to remove extraneous datafrom the beginning and end of the signal and accomplished by employing athreshold. Next, the background was removed from the signal, so that thesignal had a common baseline for all detected colors. Finally, a lowpassfilter was applied to remove high frequency noise from the signal.

To disambiguate the detected colors, a weighted matrix was calculatedand applied to the signal to amplify the color-space of thenucleotide-dye spectrum. Calculation of this color separation matrix wasaccomplished using the methods of Li et al. Electrophoresis 1999, 20,1433-1442. In this adaptation, a “m×n” color separation matrix iscalculated from correlating the “m” number of dyes utilized in the assaywith the “n” number of detector elements. The conversion of the signalfrom the detector space (PMT elements), to the dye space is performed bymatrix manipulation as follows: D=CSM×PMT, where D is the signal in dyespace for each of the m dyes, CSM is the color separation matrix, andPMT is a matrix with the signal from each of the n elements of thedetector.

Next, the peaks in the color separated signal were identified using acombination of zero-crossing filters and frequency analysis. Finally,for fragment sizing applications, the corrected traces wereallele-called to identify each fragment and to assign a fragment sizebased on a sizing standard. For DNA sequencing applications, thecorrected traces were base-called to associate one of the fournucleotides with each peak in the trace. A detailed description of basecalling can be found in Ewing et al. Genome Research, 1998, 8, 175-185,and Ewing et al., Genome Research, 1998, 8, 186-194, the disclosures ofthat are hereby incorporated by reference in their entirety.

3. Dye Labels

Dye labels attached to oligonucleotides and modified oligonucleotidescan be synthesized or obtained commercially (e.g. OperonBiotechnologies, Huntsville, Ala.). A large number of dyes (greater than50) are available for application in fluorescence excitationapplications. These dyes include those from the fluorescein, rhodamineAlexaFluor, Biodipy, Coumarin, and Cyanine dye families. Furthermore,quenchers are also available for labeling oligo sequences to minimizebackground fluorescence. Dyes with emission maxima from 410 nm (CascadeBlue) to 775 nm (Alexa Fluor 750) are available and can be used. Dyesranging between 500 nm to 700 nm have the advantage of being in thevisible spectrum and can be detected using conventional photomultipliertubes. The broad range of available dyes allows selection of dye setsthat have emission wavelengths that are spread across the detectionrange. Detection systems capable of distinguishing many dyes have beenreported for flow cytometry applications (see, Perfetto et al., Nat.Rev. Immunol. 2004, 4, 648-55; and Robinson et al., Proc of SPIE 2005,5692, 359-365).

Fluorescent dyes have peak excitation wavelengths that are typically 20to 50 nm blue-shifted from their peak emission wavelength. As a result,use of dyes over a wide range of emission wavelengths may require theuse of multiple excitation sources, with excitation wavelengths toachieve efficient excitation of the dyes over the emission wavelengthrange. Alternatively, energy transfer dyes can be utilized to enable asingle laser, with a single emission wavelength, to be used for excitingall dyes of interest. This is achieved by attaching an energy transfermoiety to the dye label. This moiety is typically another fluorescentdye with an absorption wavelength that is compatible with the excitationwavelength of the light source (e.g. laser). Placement of this absorberin close proximity with an emitter allows the absorbed energy to betransferred from the absorber to the emitter, allowing for moreefficient excitation of the long wavelength dyes (Ju et al., Proc NatlAcad Sci USA 1995, 92, 4347-51).

Dye labeled dideoxynucleuotides are available from Perkin Elmer,(Waltham, Mass.).

B. EXAMPLES Example 5 Six-Color Separation and Detection of NucleicAcids

The following example illustrates the separation and detection ofnucleic acid fragments labeled with 6 fluorescent dyes, and demonstratesthe color resolution capability of the spectrometer/multi elementexcitation/detection system. DNA fragments were labeled with 6-FAM, VIC,NED, PET dyes by applying fluorescently labeled primers in a multiplexedPCR amplification reaction. In this reaction, 1 ng of human genomic DNA(9947A) was amplified in a 25 μL reaction in according to themanufacturers recommended conditions (AmpFISTR Identifiler, AppliedBiosystems). 2.7 μL of the PCR product was removed and mixed with 0.3 μLof GS500-LIZ sizing standard (Applied Biosystems) and 0.3 μL ofHD400-ROX sizing standard. HiDi (Applied Biosystems) was added to atotal of 13 μL and the sample was inserted into the sample well of theseparation biochip and subjected to electrophoresis.

Electrophoretic separation of DNA using Genebench consists of a seriesof four operations: pre-electrophoresis, loading, injection andseparation. These operations are carried out on a microfluidic biochip,which is heated to a uniform temperature of 50° C. The biochip contains16 channel systems for separation and detection multiple, eachconsisting of an injector channel and a separation channel. DNA foranalysis is separated by electrophoretic transport of the DNA through asieving matrix along the separation channel. The separation length ofthe biochips is ranges from 160 to 180 mm.

The first step is pre-electrophoresis, which is accomplished by applyinga 160 V/cm field along the length of the channel for six (6) minutes.Separation buffer (TTE1X) is pipetted into the anode, cathode and wastewells. Samples for analysis are pipetted into the sample wells and a 175V applied from the sample well to the waste well for 18 seconds,followed by the application of 175 V across the sample and waste welland 390 V at the cathode for 72 seconds, to load the sample into theseparation channel. Injection of the sample is accomplished by applyinga 160 V/cm field along the length of the separation channel while fieldsof 50 V/cm and 40 V/cm are applied across the sample and waste wellsrespectively. Separation is continued with the injection voltageparameters for 30 min during that an optical system detects theseparating bands of DNA. The data collection rate is 5 Hz and PMT gainsare set to −800 V.

Sixteen samples containing amplified DNA were loaded for simultaneousseparation and detection. The signals from each of the 32-elements ofthe PMT were collected as a function of time to generate anelectrophoregram. The resulting electrophoregram (FIG. 17) shows peakscorresponding to the presence of a DNA fragment at theexcitation/detection window for one of the 16 lanes. Furthermore, therelative signal strength of each element of the 32-element PMT for eachpeak corresponds to the spectral content of the dye (or dyes if morethan one dye is present at the detection window) associated with the DNAfragment. FIG. 18 shows the emission spectra of the dyes detected, andthe background spectra of the substrate. The substrate backgroundspectra is subtracted from the spectra from each of the peaks.Performing this exercise results in the identification of 6 distinct dyespectra. The spectra of the 6-dyes are superimposed on the same plot. Acomparison of this data with the actual published dye spectra shows thatthe relative of the dyes are similar to the published data. This exampledemonstrates that the system is able to detect and differentiate the 6dyes in the reaction solution. The spectral output of this is used togenerate the color correction matrix and convert the signals fromdetector space to dye space representation (FIGS. 19 and 20).

Example 6 Eight Color Separation and Detection of Nucleic Acids

In this example, an 8 dye separation and detection of acids labeled withfluorescent dyes is shown. Forward and reverse primer pair sequences for8 loci are selected from the published sequences (Butler et al., JForensic Sci 2003, 48, 1054-64).

The loci selected are CSF1P0, FGA, THO1, TPDX, vWA, D3S1358, D5S818 andD7S820, although any of the loci and hence primer pairs described in thepaper can also be used in this example. Each of the forward primers forthe primer pairs is labeled with a separate fluorescent dye (OperonBiotechnologies, Huntsville, Ala.). Dyes selected for attachment to theprimers include Alexa Fluor Dyes 488, 430, 555, 568, 594, 633, 647, andTamra. Numerous other dyes are available and can also be used as labels.Each locus is amplified separately following the PCR reaction protocolsof (Butler, 2003, Id.) to yield a reaction solution with fragmentslabeled with their respective dyes. Template for PCR reaction is 1 ng ofhuman gemonic DNA (type 9947A from Promega, Madison Wis.).

Each PCR reaction was purified by cleaning up through a PCR cleanupcolumn, where primers (labeled and dye-labeled primers) and enzymes areremoved, and the PCR buffer is exchanged by the DI eluant. The resultingproduct of clean is a solution of labeled DNA fragments in DI water.Cleanup of dye labeled products follows the protocol of Smith usingMinElute™ columns (Qiagen, Valencia, Calif.). A total of eight reactionsare performed. Eight cleaned up PCR reactions were mixed together in aratio to generate peaks of equivalent signal strengths, generating amixture containing fragments labeled with 8 different dyes.Alternatively, primers for 8 loci can be mixed together to form a masterprimer mix for multiplexed amplification.

This solution is separated and detected with the instrument and protocolas described in Example 1. The grating of the spectrograph is adjustedsuch that the emission of the 8 dyes falls across the 32 pixels of thedetector elements. The amount of sample loaded for analysis is to beadjusted such that detected signals fall within the dynamic range of thedetection system.

Example 7 Spectrometer/Multi-Element PMT System

The following example illustrates separation/detection of labeled DNAfragments with the spectrometer/multi-element PMT system of FIG. 16,specifically for identifying sequence of a DNA template. In thisreaction, 0.1 pmol of DNA template M13 and M13 sequencing primer wasamplified with the GE Amersham BigDye™ sequencing kit, according to therecommended reaction conditions. The reaction mix was cleaned up byethanol precipitation and resuspended in 13 μL of DI water. The samplewas separated following the electrophoretic separation condition asdescribed in example 5. Sample loading conditions were modified and wascarried out by applying 175 V across the sample well to the waste wellfor 105 seconds. FIG. 21 shows an electrophoregram for the DNA sequence,with colored traces representing the detector element corresponding tothe spectral maximum for each of the 4 dyes used. The sequence obtainedwas base called with Phred quality score of >20 for 519 bases and QV30of 435 bases (FIG. 22).

Example 8 Simultaneous Separation and Detection of Products of TwoSequencing Reactions

In this example, separation and detection of fragments from cyclesequencing of two DNA templates are carried out simultaneously in asingle separation channel. The cycle sequencing reactions can beprepared by either dye labeled terminator reactions or dye labeledprimer reactions as follows:

For Dye Labeled Terminator Reactions:

Cycle sequencing reaction for each template fragment consisting of asequencing primer appropriate for the template sequence of interest, andreagents for conducting DNA sequencing including cycle sequencingbuffer, polymerase, oligonucleotides, dideoxynucleotides and labeleddideoxynucleotides is prepared. Eight different dyes are utilized forthe labeling. In the first cycle sequencing reaction, one set of 4 dyelabeled dideoxy nucleotides is used. In the second cycle sequencingreaction, another set of 4 dye labeled dideoxy nucleotides (withemission wavelengths different than those of the four used in the firstcycle sequencing reaction) is used. Each cycle sequencing reaction iscarried out separately following a protocol that thermally cycles eachreaction multiple times. Each thermal cycle includes a denature, annealand extension step with temperatures and times following the protocolsof Sanger (see, Sanger et al., Proc Natl Acad Sci USA 1977, 74, 5463-7).The cycle sequencing product from the two reactions are combined to forma sample that consist of labeled DNA fragments, with a total of eightunique dyes, from each of the two DNA templates.

For Dye Labeled Primer Reactions:

Alternatively, the sample for separation and detection can be fabricatedby using primer labeled cycle sequencing. Four cycle sequencingreactions are carried out for each DNA template. Each reaction is acycle sequencing reaction consisting of a labeled sequencing primer, andreagents for conducting DNA sequencing including cycle sequencingbuffer, polymerase, oligonucleotides. In addition, each reaction willinclude one of the dideoxynucleuotides (ddATP, ddTTP, ddCTP, or ddGTP)and one labeled primer. Each dye associated with the primer is unique inemission wavelength and is correlated with the type of dideoxynucleotide in the cycle sequencing solution (ddATP, ddTTP, ddCTP, orddGTP). Each cycle sequencing reaction is carried out separatelyfollowing a protocol that thermally cycles each reaction multiple times.Each thermal cycle includes a denature, anneal and extension step withtemperatures and times following the protocols of Sanger (see, Sanger,1977, Id.). For cycle sequencing the second DNA template, another set of4 dyes (with emission wavelength different to that of the four used inthe first cycle sequencing reaction) is applied. The product of alleight reactions (each with a different dye) are mixed together to form asample that consist of DNA fragments from each of the two DNA templates.

Sample for Separation and Detection:

Each of the sequencing reactions is cleaned up by ethanol precipitation.Separation and detection of the sample follows the protocol of Example8. The result of the separation and detection is the generation of twodistinct DNA sequences, corresponding to each of the two template DNAfragments.

The methods of this example can be modified to allow the use of dyes inmultiples of four to allow detection of that multiple of DNA sequencesin a single separation channel (e.g. 12 dyes for the detection of 3sequences simultaneously, 16 dyes for the detection of 4 sequencessimultaneously, 20 dyes for the detection of five sequencessimultaneously, and so on). Finally, separation of the labeled fragmentsneed not be limited to electrophoresis.

Example 9 Separation and Detection of 500 or More Loci in a SingleChannel

There are several applications of nucleic acid analysis that can beapplied to clinical diagnostics, including DNA and RNA sequencing andfragment size determination. In this example, the use of simultaneousdetection of 10 colors allows the interrogation of up to 500 loci.Analysis of the size of large number of fragments can be utilized toidentify pathogens or to characterize many loci within an individual'sgenome, for example. In the setting of prenatal and pre-implantationgenetic diagnosis, aneuploidy is currently diagnosed by karyotyping andby fluorescent in situ hybridization (FISH). In FISH studies, thepresence of two signals per cell indicates that two copies of a givenlocus are present within that cell, one signal indicates monosomy orpartial monosomy, and three signals indicates trisomy or partialtrisomy. FISH typically utilizes approximately 10 probes to assesswhether or not a cell contains a normal chromosomal complement. Thisapproach does not allow a detailed view of the entire genome, however,and cells that appear normal by FISH may well have major abnormalitiesthat are not detected by the technique.

The teachings of the present invention make use of multicolor separationand detection to allow approximately 500 chromosomal loci widelydispersed about across all chromosomes to be assessed to allow a muchmore detailed analysis of chromosomal structure. In this example, primerpair sequences for approximately 500 loci are selected from publishedsequences, with each locus present as a single copy per haploid genome.In addition, 10 sets of 50 primer pairs are selected such that each setdefines a corresponding set of DNA fragments such that none of thefragments are of the identical size. For each set, the forward primersfor the primer pairs are labeled with one fluorescent dye, and no twosets share the same dye. Dyes selected for attachment to the primers areAlexa Fluor Dyes 488, 430, 555, 568, 594, 633, 647, 680, 700, and Tamra.Numerous other dyes are available and can also be used as labels. Theloci can be amplified in one or several parallel PCR reactions asdescribed in “METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGETNUCLEIC ACIDS”, supra. The amplified primers are separated and detectedusing the methods described herein. In a single separation channel, all500 fragments can be precisely identified by size, 50 for each of tendyes.

The number of loci, dyes, and separation channels can be varied based onthe desired application. Smaller numbers of fragments can be detected ifdesired by utilizing a smaller number of dye labels or generating fewerDNA fragments per label; in this way, less than 500, less than 400, lessthan 300, less than 200, less than 100, less than 75, less than 50, lessthan 40, less than 30, or less than 20 fragments can be detected asdesired. The maximum number of loci that can be identified per lane isbased on the read length and resolution of the separation system (e.g.,single base pair resolution of DNA fragments ranging from 20 to 1500base pairs results in hundreds of fragments) multiplied by the number ofdistinct dyes that can be detected (as noted supra, dozens areavailable). Accordingly, thousands of loci can be identified in a singleseparation channel, and the number will increase as additional dyes aredeveloped.

What is claimed is:
 1. A method for multiple-sample DNA analysis,comprising: injecting a first template DNA extracted based on a firstsample from a first sample chamber on a biochip through a first channelto a first reaction reservoir in a first region of said biochip;injecting a second template DNA extracted based on a second sample froma second sample chamber on said biochip through a second inlet to asecond reaction reservoir in the first region of said biochip, thesecond sample chamber being separate from the first sample chamber;inducing thermal cycles in the first region of the biochip for PCRamplification of DNA fragments, the first region including at least thefirst reaction reservoir designated for PCR amplification based on thefirst sample, and the second reaction reservoir designated for PCRamplification based on the second sample; inducing liquid flow torespectively move first amplified DNA fragments from the first reactionreservoir to a first separation unit in a second region of the biochip,and second amplified DNA fragments from the second reaction reservoir toa second separation unit in the second region of the biochip; inducingelectric fields in the first separation unit to separate the firstamplified DNA fragments by size in a first separation channel on thebiochip; inducing electric fields in the second separation unit toseparate the second amplified DNA fragments by size in a secondseparation channel on the biochip, the second separation channel beingfluidically separated from the first separation channel; and detectingthe separated DNA fragments.
 2. The method of claim 1, furthercomprising: extracting the first template DNA from the first sample; andextracting the second template DNA from the second sample.
 3. The methodof claim 1, further comprising: injecting a third template DNA extractedbased on a third sample from a third sample chamber on a biochip througha third channel to a third reaction reservoir in a first region of saidbiochip, the third sample chamber being separate from the first andsecond sample chambers; injecting a fourth template DNA extracted basedon a fourth sample from a fourth sample chamber on said biochip througha fourth inlet to a fourth reaction reservoir in the first region ofsaid biochip, the fourth sample chamber being separate from the first,second and third sample chambers; inducing thermal cycles in the firstregion of the biochip for PCR amplification of DNA fragments, the firstregion including at least the third reaction reservoir designated forPCR amplification based on the third sample, and the fourth reactionreservoir designated for PCR amplification based on the fourth sample;inducing liquid flow to respectively move first amplified DNA fragmentsfrom the third reaction reservoir to a third separation unit in saidsecond region of the biochip, and fourth amplified DNA fragments fromthe fourth reaction reservoir to a fourth separation unit in the secondregion of the biochip; inducing electric fields in the third separationunit to separate the third amplified DNA fragments by size in a thirdseparation channel on the biochip; inducing electric fields in thefourth separation unit to separate the fourth amplified DNA fragments bysize in a fourth separation channel on the biochip, the fourthseparation channel being fluidically separated from the first, secondand third separation channels; and detecting the separated DNAfragments.
 4. The method of claim 2, further comprising: injecting firstreagents and the first template DNA into the first reaction reservoir;and injecting second reagents and the second template DNA into thesecond reaction reservoir.
 5. The method of claim 1, wherein detectingthe separated DNA fragments further comprises: one or more light sourcespositioned for illuminating a first detection position on said firstseparation channel and a second detection position on said secondseparation channel; a mirror to scan said one or more light sourcessequentially between detection positions; one or a plurality of firstoptical elements positioned for collecting and directing light emanatingfrom the detection positions; and a light detector positioned to acceptlight directed from the one or plurality of first optical elements. 6.The method of claim 5 wherein the light detector comprises a wavelengthdispersive element to disperse the light from the one or plurality offirst optical elements according to light wavelength into at least 6wavelength components and, the wavelength dispersive element ispositioned to provide at least a portion of the dispersed at least 6wavelength components to at least 6 detection elements, wherein each ofthe detection elements are in communication with a first control elementfor simultaneously collecting detection information from each of thedetection elements and, wherein said light detector detects fluorescencefrom at least 6 dyes labeled to one or more biological molecules, eachdye having a unique peak emission wavelength.
 8. The method of claim 1,wherein inducing liquid flow to respectively move the first amplifiedDNA fragments from the first reaction reservoir to the first separationunit in the second region of the biochip, and the second amplified DNAfragments from the second reaction reservoir to the second separationunit in the second region of the biochip, further comprises: inducingliquid flow to move a first PCR mixture having the first amplified DNAfragments from the first reaction reservoir to a first dilutionreservoir; diluting the first PCR mixture with a first dilutant;inducing liquid flow to move a second PCR mixture having the secondamplified DNA fragments from the second reaction reservoir to a seconddilution reservoir; and diluting the second PCR mixture with a seconddilutant.
 9. The method of claim 1: wherein inducing the electric fieldin the second separation unit to separate the second amplified DNAfragments by size in the second separation channel on the microfluidicchip further comprises: Inducing the electric fields in the secondseparation unit simultaneously as in the first separation unit tosimultaneously separate the second amplified DNA fragments and the firstamplified DNA fragments.
 10. The method of claim 5 wherein the lightsource is a single laser, the wavelength dispersive element is a prism,diffraction gradient, transmission gradient, spectrograph or holographicdiffraction grating, and each of said at least 6 detection elements is alinear multi-anode photomultiplier tube.